Isolated nucleic acid molecules coding for tumor rejection antigen precursor MAGE-2 and uses thereof

ABSTRACT

Isolated nucleic acid molecules are described, which have complements that hybridize to a nucleic acid molecule referred to as MAGE-2. The isolated nucleic acid molecules are expressed in some melanoma cells, as well as other types of cancer cells. The invention also relates to nucleic acid molecules which encode fragments of the proteins encoded by these nucleic acid molecules, and nucleic acid molecules which encode the ultimate product, the so-called tumor rejection antigens.

RELATED APPLICATION

This application is a divisional of Ser. No. 09/404,026, filed Sep. 23, 1999, which is a divisional of Ser. No. 08/967,727, filed on Nov. 27, 1997, now U.S. Pat. No. 6,025,474, which is a divisional of Ser. No. 08/037,230, filed Mar. 26, 1993, now U.S. Pat. No. 6,235,525 which is a continuation-in-part of PCT Application PCT/US92/04354 filed on May 22, 1992 designating the United States, which is a continuation-in-part of Ser. No. 07/807,043, filed Dec. 12, 1991 now U.S. Pat. No. 5,342,774, which is a continuation-in-part of Ser. No. 07/764,364, filed Sep. 23, 1991 now U.S. Pat. No. 5,327,252, which is a continuation-in-part of Ser. No. 07/728,838, filed Jul. 9, 1991 now abandoned, which is a continuation-in-part of Ser. No. 07/705,702 now abandoned, filed May 23, 1991, and now abandoned.

FIELD OF THE INVENTION

This invention relates in general to the field of immunogenetics as applied to the study of oncology. More specifically, it relates to the study and analysis of mechanisms by which tumors are recognized by the organism's immune system such as through the presentation of so-called tumor rejection antigens, and the expression of what will be referred to herein as “tumor rejection antigen precursors” or “TRAPs”. Most specifically, it refers to nucleic acid molecules coding for one such TRAP, i.e., MAGE-3, which is processed to a tumor rejection antigen or “TRA” presented by HLA-A1 molecules.

BACKGROUND AND PRIOR ART

The study of the recognition or lack of recognition of cancer cells by a host organism has proceeded in many different directions. Understanding of the field presumes some understanding of both basic immunology and oncology.

Early research on mouse tumors revealed that these displayed molecules which led to rejection of tumor cells when transplanted into syngeneic animals. These molecules are “recognized” by T-cells in the recipient animal, and provoke a cytolytic T-cell response with lysis of the transplanted cells. This evidence was first obtained with tumors induced in vitro by chemical carcinogens, such as methylcholanthrene. The antigens expressed by the tumors and which elicited the T-cell response were found to be different for each tumor. See Prehn, et al., J. Natl. Canc. Inst. 18: 769-778 (1957); Klein et al., Cancer Res. 20: 1561-1572 (1960); Gross, Cancer Res. 3: 326-333 (1943), Basombrio, Cancer Res. 30: 2458-2462 (1970) for general teachings on inducing tumors with chemical carcinogens and differences in cell surface antigens. This class of antigens has come to be known as “tumor specific transplantation antigens” or “TSTAs”. Following the observation of the presentation of such antigens when induced by chemical carcinogens, similar results were obtained when tumors were induced in vitro via ultraviolet radiation. See Kripke, J. Natl. Canc. Inst. 53: 333-1336 (1974).

While T-cell mediated immune responses were observed for the types of tumor described supra, spontaneous tumors were thought to be generally non-immunogenic. These were therefore believed not to present antigens which provoked a response to the tumor in the tumor carrying subject. See Hewitt, et al., Brit. J. Cancer 33: 241-259 (1976).

The family of tum⁻ antigen presenting cell lines are immunogenic variants obtained by mutagenesis of mouse tumor cells or cell lines, as described by Boon et al., J. Exp. Med. 152: 1184-1193 (1980), the disclosure of which is incorporated by reference. To elaborate, tum⁻ antigens are obtained by mutating tumor cells which do not generate an immune response in syngeneic mice and will form tumors (i.e., “tum⁺” cells). When these tum⁺ cells are mutagenized, they are rejected by syngeneic mice, and fail to form tumors (thus “tum⁻”) . See Boon et al., Proc. Natl. Acad. Sci. USA 74: 272 (1977), the disclosure of which is incorporated by reference. Many tumor types have been shown to exhibit this phenomenon. See, e.g., Frost et al., Cancer Res. 43: 125 (1983).

It appears that tum⁻ variants fail to form progressive tumors because they elicit an immune rejection process. The evidence in favor of this hypothesis includes the ability of “tum⁻” variants of tumors, i.e., those which do not normally form tumors, to do so in mice with immune systems suppressed by sublethal irradiation, Van Pel et al., Proc. Natl, Acad. Sci. USA 76: 5282-5285 (1979); and the observation that intraperitoneally injected tum⁻ cells of mastocytoma P815 multiply exponentially for 12-15 days, and then are eliminated in only a few days in the midst of an influx of lymphocytes and macrophages (Uyttenhove et al., J. Exp. Med. 152: 1175-1183 (1980)). Further evidence includes the observation that mice acquire an immune memory which permits them to resist subsequent challenge to the same tum⁻ variant, even when immunosuppressive amounts of radiation are administered with the following challenge of cells (Boon et al., Proc. Natl, Acad. Sci. USA 74: 272-275 (1977); Van Pel et al., supra; Uyttenhove et al., supra). Later research found that when spontaneous tumors were subjected to mutagenesis, immunogenic variants were produced which did generate a response. Indeed, these variants were able to elicit an immune protective response against the original tumor. See Van Pel et al., J. Exp. Med. 157: 1992-2001 (1983). Thus, it has been shown that it is possible to elicit presentation of a so-called “tumor rejection antigen” in a tumor which is a target for a syngeneic rejection response. Similar results have been obtained when foreign genes have been transfected into spontaneous tumors. See Fearson et al., Cancer Res. 48: 2975-1980 (1988) in this regard.

A class of antigens has been recognized which are presented on the surface of tumor cells and are recognized by cytotoxic T cells, leading to lysis. This class of antigens will be referred to as “tumor rejection antigens” or “TRAs” hereafter. TRAs may or may not elicit antibody responses. The extent to which these antigens have been studied, has been via cytolytic T cell characterization studies, in vitro i.e., the study of the identification of the antigen by a particular cytolytic T cell (“CTL” hereafter) subset. The subset proliferates upon recognition of the presented tumor rejection antigen, and the cells presenting the antigen are lysed. Characterization studies have identified CTL clones which specifically lyse cells expressing the antigens. Examples of this work may be found in Levy et al., Adv. Cancer Res. 24: 1-59 (1977); Boon et al., J. Exp. Med. 152: 118-1193 (1980); Brunner et al., J. Immunol. 124: 1627-1634 (1980); Maryanski et al., Eur. J. Immunol. 124: 1627-1634 (1980); Maryanski et al., Eur. J. Immunol. 12: 406-412 (1982); Palladino et al., Canc. Res. 47: 5074-5079 (1987). This type of analysis is required for other types of antigens recognized by CTLs, including minor histocompatibility antigens, the male specific H-Y antigens, and a class of antigens, referred to as “tum⁻” antigens, and discussed herein.

A tumor exemplary of the subject matter described supra is known as P815. See DePlaen et al., Proc. Natl. Acad. Sci. USA 85: 2274-2278 (1988); Szikora et al., EMBO J 9: 1041-1050 (1990), and Sibille et al., J. Exp. Med. 172: 35-45 (1990), the disclosures of which are incorporated by reference. The P815 tumor is a mastocytoma, induced in a DBA/2 mouse with methylcholanthrene and cultured as both an in vitro tumor and a cell line. The P815 line has generated many tum⁻ variants following mutagenesis, including variants referred to as P91A (DePlaen, supra), 35B (Szikora, supra), and P198 (Sibille, supra). In contrast to tumor rejection antigens—and this is a key distinction—the tum⁻ antigens are only present after the tumor cells are mutagenized. Tumor rejection antigens are present on cells of a given tumor without mutagenesis. Hence, with reference to the literature, a cell line can be tum⁺, such as the line referred to as “P1”, and can be provoked to produce tum⁻variants. Since the tum⁻ phenotype differs from that of the parent cell line, one expects a difference in the DNA of tum⁻ cell lines as compared to their tum⁺ parental lines, and this difference can be exploited to locate the gene of interest in tum⁻ cells. As a result, it was found that genes of tum⁻ variants such as P91A, 35B and P198 differ from their normal alleles by point mutations in the coding regions of the gene. See Szikora and Sibille, supra, and Lurquin et al., Cell 58: 293-303 (1989). This has proved not to be the case with the TRAs of this invention. These papers also demonstrated that peptides derived from the tum⁻ antigen are presented by the L^(d) molecule for recognition by CTLs. P91A is presented by L^(d), P35 by D^(d) and P198 by K^(d).

Prior patent applications PCT/US92/04354, U.S. Ser. Nos. 807,043; 764,364; 728,838 and 707,702, all of which are incorporated by reference, describe inventions involving, inter alia, genes and other nucleic acid molecules which code for various TRAPs, which are in turn processed to tumor rejection antigen, or “TRAs”.

The genes are useful as a source for the isolated and purified tumor rejection antigen precursor and the TRA themselves, either of which can be used as an agent for treating the cancer for which the antigen is a “marker”, as well as in various diagnostic and surveillance approaches to oncology, discussed infra. It is known, for example, that tum⁻ cells can be used to generate CTLs which lyse cells presenting different tum⁻ antigens as well as tum⁺ cells. See, e.g., Maryanski et al., Eur. J. Immunol 12: 401 (1982); and Van den Eynde et al., Modern Trends in Leukemia IX (June 1990), the disclosures of which are incorporated by reference. The tumor rejection antigen precursor may be expressed in cells transfected by the gene, and then used to generate an immune response against a tumor of interest.

In the parallel case of human neoplasms, it has been observed that autologous mixed lymphocyte-tumor cell cultures (“MLTC” hereafter) frequently generate responder lymphocytes which lyse autologous tumor cells and do not lyse natural killer targets, autologous EBV-transformed B cells, or autologous fibroblasts (see Anichini et al., Immunol. Today 8: 385-389 (1987)). This response has been particularly well studied for melanomas, and MLTC have been carried out either with peripheral blood cells or with tumor infiltrating lymphocytes. Examples of the literature in this area including Knuth et al., Proc. Natl. Acad. Sci. USA 86: 2804-2802 (1984); Mukherji et al., J. Exp. Med. 158: 240 (1983); Hérin et all, Int. J. Canc. 39: 390-396 (1987); Topalian et al, J. Clin. Oncol 6: 839-853 (1988). Stable cytotoxic T cell clones (“CTLs” hereafter) have been derived from MLTC responder cells, and these clones are specific for the tumor cells. See Mukherji et al., supra, Herin et all, supra, Knuth et al., supra. The antigens recognized on tumor cells by these autologous CTLs do not appear to represent a cultural artifact, since they are found on fresh tumor cells. Topalian et al., supra; Degiovanni et al., Eur. J. Immunol. 20: 1865-1868 (1990). These observations, coupled with the techniques used herein to isolate the genes for specific murine tumor rejection antigen precursors, have led to the isolation of nucleic acid sequences coding for tumor rejection antigen precursors of TRAs presented on human tumors. It is now possible to isolate the nucleic acid sequences which code for tumor rejection antigen precursors, including, but not being limited to those most characteristic of a particular tumor, with ramifications that are described infra.

Additional work has focused upon the presentation of TRAs by the class of molecules known as human leukocyte antigens, or “HLAS”. This work has resulted in several unexpected discoveries regarding the field. Specifically in U.S. patent application Ser. No. 938,334, the disclosure of which is incorporated by reference, nonapeptides are taught which are presented by the HLA-A1 molecule. The reference teaches that given the known specificity of particular peptides for particular HLA molecules, one should expect a particular peptide to bind one HLA molecule, but not others. This is important, because different individuals possess different HLA phenotypes. As a result, while identification of a particular peptide as being a partner for a specific HLA molecule has diagnostic and therapeutic ramifications, these are only relevant for individuals with that particular HLA phenotype. There is a need for further work in the area, because cellular abnormalities are not restricted to one particular HLA phenotype, and targeted therapy requires some knowledge of the phenotype of the abnormal cells at issue.

In U.S. patent application Ser. No. 008,446, filed Jan. 22, 1993 and incorporated by reference, the fact that the MAGE-1 expression product is processed to a second TRA is disclosed. This second TRA is presented by HLA-C10-molecules. The disclosure shows that a given TRAP can yield a plurality of TRAs.

In U.S. patent application Ser. No. 994,928, filed Dec. 22, 1992, and incorporated by reference herein, tyrosinase is described as a tumor rejection antigen precursor. This reference discloses that a molecule which is produced by some normal cells (e.g., melanocytes), is processed in tumor cells to yield a tumor rejection antigen that is presented by HLA-A2 molecules.

It was mentioned, supra, that different individuals possess different HLA types. It has also been found that the expression of particular MAGE genes is not always linked to particular disorders, or individuals of particular HLA types. Thus, one cannot state, e.g., that all melanoma patients will express MAGE-1 TRAP nor could one say categorically that MAGE-1 expression is limited to melanoma patients of type HLA-A1. Further, one cannot state that only one type of TRAP is expressed in individuals of a particular HLA type. No rules or guidelines can be pointed to which correlate any of these factors.

Thus, it is not expected that a second TRAP is processed to a TRAP which is presented by HLA-A1 molecules. It has now been found that in addition to MAGE-1, a TRA derived from MAGE-3 TRAP is presented by HLA-A1 molecules. This is shown in examples 37-40, which follow, together with a discussion of the ramifications of this discovery.

These and various other aspects of the invention are elaborated upon in the disclosure which follows.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B depicts detection of transfectants expressing antigen P815A.

FIG. 2 shows the sensitivity of clones P1.HTR, PO.HTR, genomic transfectant P1A.T2 and cosmid transfectant P1A.TC3.1 to lysis by various CTLs, as determined by chromium release assays.

FIG. 3 is a restriction map of cosmid C1A.3.1.

FIG. 4 shows Northern Blot analysis of expression of gene P1A.

FIG. 5 sets forth the structure of gene P1A with its restriction sites.

FIG. 6 shows the results obtained when cells were transfected with the gene from P1A, either isolated from P815 or normal cells and then tested with CTL lysis.

FIG. 7 shows lytic studies using mast cell line L138. 8A.

FIG. 8 is a map of subfragments of the 2.4 kb antigen E fragment sequence which also express the antigen.

FIG. 9 shows homology of sections of exon 3 from genes mage 1, 2 and 3.

FIG. 10 shows the result of Northern blots for MAGE genes on various tissues.

FIG. 11 presents the data of FIG. 13 in table form.

FIG. 12 shows Southern Blot experiments using the various human melanoma cell lines employed in this application.

FIG. 13 is a generalized schematic of the expression of MAGE 1, 2 and 3 genes by tumor and normal tissues.

FIG. 14 shows results from a chromium release assay using CTL clone 20/38 on various cell lines.

FIG. 15 presents the result of assays undertaken to determine antigenic specificity of CTL clone 20/38.

FIG. 16 shows the results obtained when a TNF release assay was carried out on various transfected cells.

BRIEF DESCRIPTION OF SEQUENCES

SEQ ID NO: 1 is cDNA for part of gene P1A.

SEQ ID NO: 2 presents coding region of cDNA for gene P1A.

SEQ ID NO: 3 shows non coding DNA for P1A cDNA which is 3′ to the coding region of SEQ ID NO: 2.

SEQ ID NO: 4 is the entire sequence of cDNA for P1A.

SEQ ID NO: 5 is the genomic DNA sequence for P1A.

SEQ ID NO: 6 shows the amino acid sequence for the antigenic peptides for P1A TRA. The sequence is for cells which are A⁺ B⁺, i.e., express both the A and B antigens.

SEQ ID NO: 7 is a nucleic acid sequence coding for antigen E.

SEQ ID NO: 8 is a nucleic acid sequence coding for MAGE-1.

SEQ ID NO: 9 is the gene for MAGE-2.

SEQ ID NO: 10 is the gene for MAGE-21.

SEQ ID NO: 11 is cDNA for MAGE-3.

SEQ ID NO: 12 is the gene for MAGE-31.

SEQ ID NO: 13 is the gene for MAGE-4.

SEQ ID NO: 14 is the gene for MAGE-41.

SEQ ID NO: 15 is cDNA for MAGE-4.

SEQ ID NO: 16 is cDNA for MAGE-5.

SEQ ID NO: 17 is genomic DNA for MAGE-51.

SEQ ID NO: 18 is cDNA for MAGE-6.

SEQ ID NO: 19 is genomic DNA for MAGE-7.

SEQ ID NO: 20 is genomic DNA for MAGE-8.

SEQ ID NO: 21 is genomic DNA for MAGE-9.

SEQ ID NO: 22 is genomic DNA for MAGE-10.

SEQ ID NO: 23 is genomic DNA for MAGE-11.

SEQ ID NO: 24 is genomic DNA for smage-I.

SEQ ID NO: 25 is genomic DNA for smage-II.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Many different “MAGE” genes have been identified, as will be seen from the sequences which follow the application. The protocols described in the following examples were used to isolate these genes and cDNA sequences.

“MAGE” as used herein refers to a nucleic acid sequence isolated from human cells. The acronym “smage” is used to describe sequences of murine origin.

When “TRAP” or “TRAs” are discussed herein as being specific to a tumor type, this means that the molecule under consideration is associated with that type of tumor, although not necessarily to the exclusion of other tumor types.

EXAMPLE 1

In order to identify and isolate the gene coding for antigen P815A, gene transfection was used. This approach requires both a source of the gene, and a recipient cell line. Highly transfectable cell line P1.HTR was the starting material for the recipient, but it could not be used without further treatment, as it presents “antigen A”, one of four recognized P815 tumor antigens. See Van Pel et al., Molecular Genetics 11: 467-475 (1985) . Thus, screening experiments were carried out to isolate cell lines which did not express the antigen and which nonetheless possessed P1.HTR's desirable qualities.

To do this, P1.HTR was screened with CTLs which were specific for each of tumor antigens A, B, C and D. Such CTLs are described by Uyttenhove et al., J. Exp. Med. 157: 1040-1052 (1983).

To carry out the selection, 10⁶ cells of P1.HTR were mixed with 2-4×10⁶ cells of the CTL clone in a round bottom tube in 2 ml of medium, and centrifuged for three minutes at 150×g. After four hours at 37° C., the cells were washed and resuspended in 10 ml of medium, following Maryanski et al., Eur. J. Immunol. 12: 406-412 (1982). Additional information on the CTL assay and screening protocol, in general may be found in Boon et al., J. Exp. Med. 152: 1184-1193 (1980), and Maryanski et al., Eur. J. Immunol. 12: 406-412 (1982), the disclosure of which are incorporated by reference.

When these selections were carried out, a cell line variant was found which expressed neither antigen A or B. Additional selections with CTLs specific for antigen C then yielded a variant which also lacked antigen C. Please see FIG. 2 for a summary of the results of these screenings. The variant PO.HTR is negative for antigens A, B and C, and was therefore chosen for the transfection experiments.

The cell line PO.HTR has been deposited in accordance with the Budapest Treaty at the Institute Pasteur Collection Nationale De Cultures De Microorganismes, 28, Rue de Docteur Roux, 75724 Paris France, and has accession number I-1117.

This methodology is adaptable to secure other cell lines which are variants of a cell type which normally presents at least one of the four recognized P815 tumor antigens, i.e., antigens A, B, C and D, where the variants present none of antigens A, B and C. P1.HTR is a mastocytoma cell line, so it will be seen that the protocol enables the isolation of biologically pure mastocytoma cell lines which express none of P815 antigens A, B and C, but which are highly transfectable. Other tumor types may also be screened in this fashion to secure desired, biologically pure cell lines. The resulting cell lines should be at least as transfectable with foreign DNA as is P1.HTR, and should be selected so as to not express a specific antigen.

EXAMPLE 2

Previous work reported by DePlaen et al., Proc. Natl. Acad. Sci. USA 85: 2274-2278 (1988) the disclosure of which is incorporated by reference herein had shown the efficacy of using cosmid library transfection to recover genes coding for tum⁻ antigens.

Selective plasmid and genomic DNA of P1.HTR were prepared, following Wölfel et al., Immunogenetics 26: 178-187 (1987). The transfection procedure followed Corsaro et al., Somatic Cell Molec. Genet 7: 603-616 (1981), with some modification. Briefly, 60 μg of cellular DNA and 3 μg of DNA of plasmid pHMR272, described by Bernard et al., Exp. Cell. Biol. 158: 237-243 (1985) were mixed. This plasmid confers hygromycin resistance upon recipient cells, and therefore provides a convenient way to screen for transfectants. The mixed DNA was combined with 940 ul of 1 mM Tris-HCl (pH 7.5), 0.1 mM EDTA; and 310 ul 1M CaCl₂. The solution was added slowly, and under constant agitation to 1.25 ml of 50 mM Hepes, 280 mM NaCl, 1.5 mM Na₂HPO₄, adjusted to pH 7.1 with NaOH. Calcium phosphate—DNA precipitates were allowed to form for 30-45 minutes at room temperature. Following this, fifteen groups of PO.HTR cells (5×10⁶) per group were centrifuged for 10 minutes at 400 g. Supernatants were removed, and pellets were resuspended directly into the medium containing the DNA precipitates. This mixture was incubated for 20 minutes at 37° C., after which it was added to an 80 cm² tissue culture flask containing 22.5 ml DMEM, supplemented with 10% fetal calf serum. After 24 hours, medium was replaced. Forty-eight hours after transfection, cells were collected and counted. Transfected cells were selected in mass culture using culture medium supplemented with hygromycin B (350 ug/ml). This treatment selected cells for hygromycin resistance.

For each group, two flasks were prepared, each containing 8×10⁶ cells in 40 ml of medium. In order to estimate the number of transfectants, 1×10⁶ cells from each group were plated in 5 ml DMEM with 10% fetal calf serum (FCS), 0.4% bactoagar, and 300 ug/ml hygromycin B. The colonies were then counted 12 days later. Two independent determinations were carried out and the average taken. This was multiplied by 5 to estimate the number of transfectants in the corresponding group. Correction had to be made for the cloning efficiency of P815 cells, known to be about 0.3.

EXAMPLE 3

Eight days after transfection as described in example 2, supra, antibiotic resistant transfectants were separated from dead cells, using density centrifugation with Ficoll-Paque. These cells were maintained in non-selective medium for 1 or 2 days. The cells were plated in 96 well microplates (round bottom), at 30 cells/microwell in 200 ul of culture medium. Anywhere from 100-400 microwells were prepared, depending on the number of transfectants prepared. Agar colony tests gave estimates of 500-3000. After 5 days, the wells contained about 6×10⁴ cells and replicate plates were prepared by transferring {fraction (1/10)} of the wells to microplates which were then incubated at 30° C. One day later, master plates were centrifuged, medium removed, and 750 CTLs against P815 antigen A (CTL-P1:5) were added to each well together with 10⁶ irradiated syngeneic feeder spleen cells in CTL culture medium containing 40 U/ml recombinant human IL-2, and HAT medium to kill stimulator cells. Six days later, plates were examined visually to identify wells where CTLs had proliferated. Where plates showed proliferating microcultures, aliquots of 100 ul of the wells were transferred to another plate containing ⁵¹Cr labeled P1.HTR target cells (2×10³-4×10³ per well), and chromium release was measured after 4 hours. Replicate microcultures corresponding to those showing high CTL activity were expanded and cloned by limited dilution in DMEM with 10% FCS. Five days later, about 200 clones were collected and screened with the CTL.P1:5 cell line, described supra, in a visual lysis assay. See FIG. 1A for these results.

In these experiments, three of the fifteen groups of transfectants yielded a few positive microcultures. These microcultures were tested for lytic activity against P1.HTR, as described supra. Most of the microcultures where proliferation was observed showed lytic activity. This activity was well above background, as shown in FIG. 1B. This figure summarizes data wherein two groups of cells (groups “5” and “14”), 400 and 300 microwells were seeded with 30 hygromycin resistant transfected cells. Amplification and duplication of the microcultures was followed by addition of anti-A CTL P1:5. Six days later, lytic activity against P1.HTR was tested. In the figure, each point represents lytic activity of a single microculture.

Duplicate microcultures corresponding to several positive wells were subcloned, and more than 1% of the subclones were found to be lysed by anti-A CTL. Thus, three independent transfectants expressing P815A were obtained from 33,000 hygromycin resistant transfectants. One of these lines, referred to hereafter as P1A.T2 was tested further.

The relevant antigen profile of P1A.T2 is shown in FIG. 2, this being obtained via anti-CTL assays of the type described supra.

EXAMPLE 4

The CTL assays carried out for P1A.T2 demonstrated that it presented antigen A (“P815A”), and therefore had received the gene from P1.HTR. To that end, this cell line was used as a source for the gene for the antigen precursor in the following experiments.

Prior work had shown that genes coding for tum⁻ antigens could be recovered directly from transfectants obtained with a cosmid library. See DePlaen et al., Proc. Natl. Acad. Sci. USA 85: 2274-2278 (1988). This procedure was followed for recovery of the P815 gene.

Total genomic DNA of P1A.T2 was partially digested with restriction endonuclease Sau 3A1, and fractionated by NaCl density gradient ultracentrifugation to enrich for 35-50 kb DNA fragments, following Grosveld et al., Gene 10: 6715-6732 (1982). These fragments were ligated to cosmid arms of C2RB, described by Bates et al., Gene 26: 137-146 (1983), the disclosure of which is incorporated by reference. These cosmid arms had been obtained by cleavage with SmaI and treatment with calf intestinal phosphatase, followed by digestion with BamHI. Ligated DNA was packaged into lambda phage components, and titrated on E. coli ED 8767, following Grosveld et al., supra. Approximately 9×10⁵ ampicillin resistant colonies were obtained per microgram of DNA insert.

The cosmid groups were amplified by mixing 30,000 independent cosmids with 2 ml of ED 8767 in 10 mM MgCl₂, incubated 20 minutes at 37° C., diluted with 20 ml of Luria Bertani (“LB”) medium, followed by incubation for one hour. This suspension was titrated and used to inoculate 1 liter of LB medium in the presence of ampicillin (50 ug/ml). At a bacterial concentration of 2×10⁸ cells/ml (OD₆₀₀=0.8), a 10 ml aliquot was frozen, and 200 ug/ml chloramphenicol was added to the culture for overnight incubation. Total cosmid DNA was isolated by alkaline lysis procedure, and purified on CsCl gradient.

In these experiments, a library of 650,000 cosmids was prepared. The amplification protocol involved the use of 21 groups of approximately 30,000 cosmids.

EXAMPLE 5

Using the twenty-one groups of cosmids alluded to supra, (60 ug) and 4 ug of pHMR272, described supra, groups of 5×10⁶ PO.HTR cells were used as transfectant hosts. Transfection was carried out in the same manner as described in the preceding experiments. An average of 3000 transfectants per group were tested for antigen presentation, again using CTL assays as described. One group of cosmids repeatedly yielded positive transfectants, at a frequency of about 1/5,000 drug resistant transfectants. The transfectants, as with P1A.T2, also showed expression of both antigen A and B. The pattern of expression of transfectant P1A.TC3.1 is shown in FIG. 2.

EXAMPLE 6

As indicated in Example 5, supra, three independent cosmid transfected cells presenting P815A antigen were isolated. The DNA of these transfectants was isolated and packaged directly with lambda phage extracts, following DePlaen et al., Proc. Natl. Acad. Sci. USA 85: 2274-2278 (1988). The resulting product was titrated on E. coli ED 8767 with ampicillin selection, as in Example 5. Similarly, amplification of the cosmids and transfection followed Example 5, again using PO.HTR.

High frequencies of transfection were observed, as described in Table 1, which follows:

TABLE 1 Transfer of the expression of antigen P815A by cosmids obtained by direct packaging Transfectant No. of transfectants obtained with No. of cosmids obtained expressing P815A/ the cosmid by direct packaging of no. of HmB^(r) library 0.5 μg of DNA transfectants TC3.1 32 87/192 TC3.2 32000 49/384 TC3.3 44 25/72 

The cosmids were analyzed with restriction enzymes and it was found that directly packaged transfectant P1A.TC3.1 contained 32 cosmids, 7 of which were different. Each of these 7 cosmids was transfected into PO.HTR, in the manner described supra, and again, following the protocols described above, transfectants were studied for presentation of P815A. Four of the cosmid transfectants showed P815A presentation and, as with all experiments described herein, P815 B was co-expressed.

Of the four cosmids showing presentation of the two antigens, cosmid C1A.3.1 was only 16.7 kilobases long, and was selected for further analysis as described infra.

The cosmid C1A.3.1 was subjected to restriction endonuclease analysis, yielding the map shown in FIG. 3.

All EcoRI fragments were transfected, again using the above described protocols, and only the 7.4 kilobase fragment produced a transfectant that anti-A CTLs could lyse. Similar experiments were carried out on the PstI fragments, and only a 4.1 kb fragment fully contained within the 7.4 kb EcoRI fragment produced lysable transfectants.

This fragment (i.e., the 4.1 kb PstI fragment), was digested with SmaI, giving a 2.3 kb fragment which also yielded host cells presenting antigens A and B after transfection. Finally, a fragment 900 bases long, secured with SmaI/XbaI, also transferred expression of the precursors of these two antigens, i.e., the transfected host cell presented both antigen A and antigen B.

EXAMPLE 7

The 900 base fragment described above was used as a probe to detect the expression of the P815A gene in parent cell line P1.HTR. To accomplish this, total cellular RNA was first isolated using the guanidine-isothiocyanate procedure of Davis et al., Basic Methods In Molecular Biology (Elseview Science Publishing Co, New York) (1986). The same reference was the source of the method used to isolate and purify polyA⁺ mRNA using oligodT cellulose column chromatography.

Samples were then subjected to Northern Blot analysis. RNA samples were fractionated on 1% agarose gels containing 0.66 M formaldehyde. The gels were treated with 10×SSC (SSC: 0.15 M NaCl; 0.015 M sodium citrate, pH 7.0) for 30 minutes before overnight blotting on nitrocellulose membranes. These were baked for two hours at 80° C., after which the membranes were prehybridized for 15 minutes at 60° C. in a solution containing 10% dextran sulfate, 1% SDS and 1M NaCl. Hybridization was then carried out using denatured probe (the 900 base fragment), together with 100 ug/ml salmon sperm DNA.

When this protocol was carried out using P1.HTR poly A⁺ RNA, a band of 1.2 kb and two fainter bands were identified, as shown in FIG. 4, lane 1 (6 ug of the RNA).

The same probe was used to screen a cDNA library, prepared from poly-A⁺ RNA from the cell line. This yielded a clone with a 1 kb insert, suggesting a missing 5′ end. The Northern blots for the cDNA are not shown.

Hybridization experiments in each case were carried out overnight at 60° C. The blots were washed twice at room temperature with 2×SSC and twice at 60° C. with 2×SSC supplemented with 1% SDS.

The foregoing experiments delineated the DNA expressing the P815A antigen precursor sufficiently to allow sequencing, using the well known Sanger dideoxy chain termination method. This was carried out on clones generated using a variety of restriction endonucleases and by specific priming with synthetic oligonucleotide primers. The results for exons of the gene are set forth in sequence id no: 4.

EXAMPLE 8

The Northern analysis described supra suggested that the 5′ end of the cDNA was missing. To obtain this sequence, cDNA was prepared from P1.HTR RNA using a primer corresponding to positions 320-303. The sequence was then amplified using the polymerase chain reaction using a 3′ primer corresponding to positions 286-266 and a 5′ primer described by Frohman et al., Proc. Natl. Acad. Sci. USA 85: 8998-9002 (1988). A band of the expected size (270 bases) was found, which hybridized to the 900 bp SmaI/XbaI fragment described supra on a Southern blot. Following cloning into m13tg 130 λtg 131, the small, 270 bp fragment was sequenced. The sequence is shown in sequence id no: 1.

EXAMPLE 9

Following the procurement of the sequences described in Examples 7 and 8 and depicted in seq id no: 4, a 5.7 kb region of cosmid C1A.3.1 was sequenced. This fragment was known to contain the 900 base fragment which expressed P815A in transfectants. This experiment permitted delineation of introns and exons, since the cosmid is genomic in origin.

The delineated structure of the gene is shown in FIG. 5. Together with seq id no: 4, these data show that the gene for the antigen precursor, referred to as “P1A” hereafter, is approximately 5 kilobases long and contains 3 exons. An ORF for a protein of 224 amino acids starts in exon 1, ending in exon 2. The 900 base pair fragment which transfers expression of precursors for antigens A and B only contains exon 1. The promoter region contains a CAAT box, as indicated in seq. id no: 1, and an enhancer sequence. This latter feature has been observed in promoters of most MHC class I genes, as observed by Geraghty et al., J. Exp. Med 171: 1-18 (1990); Kimura et al., Cell 44: 261-272 (1986).

A computer homology search was carried out, using program FASTA with K-triple parameters of 3 and 6, as suggested by Lipman et al., Science 227: 1435-1441 (1985), and using Genbank database release 65 (October 1990). No homology was found except for a stretch of 95 bases corresponding to part of an acid region coded by exon 1 (positions 524-618), which is similar to sequences coding for acidic regions in mouse nucleolar protein NO38/B23, as described by Bourbon et al., Mol. Biol. 200: 627-638 (1988), and Schmidt-Zachmann et al., Chromosoma 96: 417-426 (1988). Fifty six of 95 bases were identical. In order to test whether these homologies were the reason for cross hybridizing, experiments were carried out using a mouse spleen cDNA library screened with the 900 base fragment. cDNA clones corresponding closely to the sizes of the cross hybridizing bands were obtained. These were partially sequenced, and the 2.6 kb cDNA was found to correspond exactly to reported cDNA sequence of mouse nucleolin, while the 1.5 kb cDNA corresponded to mouse nucleolar protein NO38/B23.

Analysis of the nucleotide sequence of the gene, referred to as “P1A” hereafter, suggests that its coded product has a molecular mass of 25 kd. Analysis of the sequence id no: 4 shows a potential nuclear targeting signal at residues 5-9 (Dingwall et al., Ann. Rev. Cell Biol. 2: 367-390 (1986)), as well as a large acidic domain at positions 83-118. As indicated supra, this contains the region of homology between P1A and the two nucleolar proteins. A putative phosphorylation site can be found at position 125 (serine). Also, a second acidic domain is found close to the C-terminus as an uninterrupted stretch of 14 glutamate residues. A similar C-terminal structure has been found by Kessel et al. Proc. Natl. Acad. Sci. USA 84: 5306-5310 (1987), in a murine homeodomain protein having nuclear localization.

In studies comparing the sequence of gene P1A to the sequences for P91A, 35B and P198, no similarities were found, showing that P1A is indicative of a different class of genes and antigens.

EXAMPLE 10

With the P1A probe and sequence in hand, investigations were carried out to determine whether the gene present in normal tissue was identical to that expressed by the tumor. To do this, phage libraries were prepared, using lambda zapII 10 and genomic DNA of DBA2 murine kidney cells. P1A was used as a probe. Hybridization conditions were as described supra, and a hybridizing clone was found. The clone contained exons one and two of the P1A gene, and corresponded to positions 0.7 to 3.8 of FIG. 5. Following localization of this sequence, PCR amplification was carried out to obtain the sequence corresponding to 3.8 to 4.5 of FIG. 5.

Sequence analysis was carried out, and no differences were found between the gene from normal kidneys and the P1A gene as obtained from the P815 tumor cells.

In further experiments, the gene as found in DBA/2 kidney cells was transfected into PO.HTR, as described supra. These experiments, presented pictorially in FIG. 7, showed that antigens A and B were expressed as efficiently by the kidney gene isolated from normal kidney cells as with the P1A gene isolated from normal kidney cells.

These experiments lead to the conclusion that the gene coding for the tumor rejection antigen precursor is a gene that does not result from a mutation; rather, it would appear that the gene is the same as one present in normal cells, but is not expressed therein. The ramifications of this finding are important, and are discussed infra.

In studies not elaborated upon herein, it was found that variants of the gene were available. Some cells were “P1A⁻B⁺”, rather than the normal “P1A”. The only difference between these is a point mutation in exon 1, with the 18th triplet coding for Ala in the variant instead of Val.

EXAMPLE 11

Additional experiments were carried out with other cell types. Following the protocols described for Northern blot hybridizations supra, RNA of normal liver and spleen cells was tested to determine if a transcript of the P1A gene could be found. The Northern blot data are presented in FIG. 4 and, as can be seen, there is no evidence of expression.

The murine P815 cell line from which P1A was isolated is a mastocytoma. Therefore, mast cell lines were studied to determine if they expressed the gene. Mast cell line MC/9, described by Nabel et al., Cell 23: 19-28 (1981), and short term cultures of bone marrow derived mast cells were tested in the manner described supra (Northern blotting), but no transcript was found. In contrast when a Balb/C derived IL-3 dependent cell line L138.8A (Hültner et al., J. Immunol. 142: 3440-3446 (1989)) was tested, a strong signal was found. The mast cell work is shown in FIG. 4.

It is known that both BALB/C and DBA/2 mice share H-² d haplotype, and thus it was possible to test sensitivity to lysis using the CTLs described supra. FIG. 8 shows these results, which essentially prove that anti-A and anti-B CTLs lysed the cells strongly, whereas anti-C and anti-D lines did not.

Further tests were carried out on other murine tumor cell lines, i.e., teratocarcinoma cell line PCC4 (Boon et al., Proc. Natl. Acad. Sci. USA 74: 272-275 (1977), and leukemias LEC and WEH1-3B. Expression could not be detected in any of these samples.

EXAMPLE 12

The actual presentation of the P1A antigen by MHC molecules was of interest. To test this, cosmid C1A.3.1 was transfected into fibroblast cell line DAP, which shows phenotype H-2^(k). The cell lines were transfected with genes expressing one of the K^(d), D^(d), and L^(d) antigen. Following transfection with both the cosmid and the MHC gene, lysis with CTLs was studied, again as described supra. These studies, summarized in Table 2, show that L^(d) is required for presentation of the P1A antigens A and B.

TABLE 2 H-2-restriction of antigens P815A and P815B No. of clones lysed by the CTL/ no. of HmB^(r) clones^(≠) Recipient cell* CTL anti-A CTL anti-B DAP (H-2^(k)) 0/208 0/194 DAP + K^(d) 0/165 0/162 DAP + D^(d) 0/157 0/129 DAP + L^(d) 25/33  15/20  *Cosmid C1A.3.1 containing the entire P1A gene was transfected in DAP cells previously transfected with H.2^(d) class I genes as indicated. ^(≠)Independent drug-resistant colonies were tested for lysis by anti-A or anti-B CTL in a visual assay.

The observation that one may associate presentation of a tumor rejection antigen with a particular MHC molecule was confirmed in experiments with human cells and HLA molecules, as elaborated upon infra.

EXAMPLE 13

Using the sequence of the P1A gene as well as the amino acid sequence derivable therefrom, antigenic peptides which were A⁺ B⁺ (i.e., characteristic of cells which express both the A and B antigens), and those which are A⁻B⁺ were identified. The peptide is presented in FIG. 10. This peptide when administered to samples of PO.HTR cells in the presence of CTL cell lines specific to cells presenting it, led to lysis of the PO.HTR cells, lending support to the view that peptides based on the product expressed by the gene can be used as vaccines.

EXAMPLE 14

The human melanoma cell line referred to hereafter as MZ2-MEL is not a clonal cell line. It expresses four stable antigens recognized by autologous CTLs, known as antigens “D, E, F, and A”. In addition, two other antigens “B” and “C” are expressed by some sublines of the tumor. CTL clones specific for these six antigens are described by van den Eynde et al., Int. J. Canc. 44: 634-640 (1989). Among the recognized subclones of MZ2-MEL are MEL.43, MEL3.0 and MEL3.1. (Van den Eynde et al., supra). Cell line MEL3.1 expresses antigen E, as determined by CTL studies as described for P815 variants, supra, so it was chosen as a source for the nucleic acid sequence expressing the antigen precursor.

In isolating the pertinent nucleic acid sequence for a tumor rejection antigen precursor, the techniques developed supra, showed that a recipient cell is needed which fulfills two criteria: (i) the recipient cell must not express the TRAP of interest under normal conditions, and (ii) it must express the relevant class I HLA molecule. Also, the recipient cell must have a high transfection frequency, i.e., it must be a “good” recipient.

In order to secure such a cell line, the clonal subline ME3.1 was subjected to repeated selection with anti-E CTL 82/30 as described by Van den Eynde, supra. The repeated cycles of selection led to isolation of subclone MZ2-MEL-2.2 isc E⁻. This subclone is also HPRT⁻, (i.e., sensitive to HAT medium: 10⁻⁴ M hypoxanthine, 3.8×10⁻⁷ aminopterine, 1.6×10⁻⁵ M 2-deoxythymidine). The subclone is referred to as “MEL-2.2” for simplicity hereafter.

EXAMPLE 15

The genomic DNA of MEL3.0 was prepared following Wölfel et al., Immunogenetics 26: 178-187 (1987), the disclosure of which is incorporated by reference. The plasmid pSVtkneoβ, as described by Nicolas et al., Cold Spring Harb., Conf. Cell Prolif. 10: 469-485 (1983) confers geneticin resistance, so it can be used as a marker for cotransfection, as it was in this experiment.

Following a procedure similar but not identical to that of Corsao et al., Somatic Cell Molec. Genet 7: 603-616 (1981), total genomic DNA and the plasmid were cotransfected. The genomic DNA (60 μg) and plasmid DNA (6 μg) were mixed in 940 μg l of 1 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, after which 310 μl of 1M CaCl₂ was added. This solution was slowly added, under constant agitation, to 1.25 ml of 2×HBS (50 mM HEPES, 280 mM NaCl 1.5 mM Na₂HPO₄, adjusted to pH 7.1 with NaOH). The calcium phosphate DNA precipitates were allowed to form for 30-45 minutes at room temperature, after which they were applied to 80 cm² tissue culture flasks which had been seeded 24 hours previously with 3×10⁶ MEL2.2 cells, in 22.5 ml of melanoma culture medium (Dulbecco's Modified Eagle's Medium) supplemented with 10% fetal calf serum. After 24 hours, the medium was replaced. Forty eight hours after transfection, the cells were harvested and seeded at 4×10⁶ cells per 80 cm² flask in melanoma culture medium supplemented with 2 mg/ml of geneticin. The geneticin serves as a selection marker.

EXAMPLE 16

Thirteen days after transfection, geneticin-resistant colonies were counted, harvested, and cultured in nonselective medium for 2 or 3 days. Transfected cells were then plated in 96-well microplates at 200 cells/well in 200 ul of culture medium with 20% fetal calf serum (FCS) in order to obtain approximately 30 growing colonies per well. The number of microcultures was aimed at achieving redundancy, i.e., such that every independent transfectant should be represented at least four times.

After 10 days, wells contained approximately 6×10⁴ cells. These cells were detached, and ⅓ of each microculture was transferred to a duplicate plate. After 6 hours, i.e., after readherence, medium was removed and 1500 anti-E CTL (CTL 82/30) , were added to each well in 100 μl of CTL culture medium with 35 U/ml of IL-2. One day later, the supernatant (50 μl) was harvested and examined for TNF concentration, for reasons set forth in the following example.

EXAMPLE 17

The size of the mammalian genome is 6×10⁶ kb. As the average amount of DNA integrated in each drug-resistant transfectant was expected to be about 200 kb, a minimum of 30,000 transfectants would need to be examined to ascertain whether antigen E had been transfected. Prior work with murine cells had shown that when a CTL stimulation assay was used, groups containing only 3% of cells expressing the antigen of interested could be identified. This should reduce the number of assays by a factor of 30. While an anti-E CTL assay, as described supra, in mixed E⁺/E⁻ cells was helpful, it was not sufficient in that consistent results could not be obtained.

As a result, an alternative test was devised. Stimulation of CTLs was studied by release of tumor necrosis factor (“TNF”) using well known methodologies which need not be repeated here. As described in Example 15, 1500 CTL 82/30 cells had been added per well of transfectants. These CTLs were collected 6 days after stimulation. As indicated supra, after ⅓ of the cells in each well had been removed and the remaining ⅔ (4×10⁴) had readhered, the CTLs and IL-2 were added thereto. The 50 μl of supernatant was removed 24 hours later and transferred to a microplate containing 3×10⁴ W13 (WEHI-164 clone 13; Espevik et al., J. Immunol. Meth. 95: 99-105 (1986)) cells in 50 μl of W13 culture medium (RPMI-1640, supplemented with L-arginine (116 mg/l), L-asparagine (36 mg/l), L-glutamine (216 mg/l), and 10% FCS supplemented with 2 μg of actinomycin D at 37% in an 8% CO₂ atmosphere. The cell line W13 is a mouse fibrosarcoma cell line sensitive to TNF. Dilutions of recombinant TNF-β in RPMI 1640 were added to target cell controls.

The W13 cultures were evaluated after 20 hours of incubation, and dead cell percentage was measured using an adaptation of the calorimetric assay of Hansen et al., J. Immunol. Meth. 119: 203-210 (1989). This involved adding 50 ml of (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide at 2.5 mg/ml in PBS, followed by two hours of incubation at 37° C. Dark blue formazan crystals were dissolved by adding 100 μl of lysis solution (1 volume N,N dimethyl formamide mixed at 37° C. with two volumes of water containing 30% (w/v) sodium dodecyl sulphate, at pH 4.7 from 1.6% acetic acid and 2.5% 1N HCl). Plates were incubated at 37° C. overnight, and ODs were taken at 570 nm using 650 nm as control. Dead cell percentage was determined via the formula: $100 \times \left\lbrack {1 - \frac{100 - \left( {{OD}_{570}\quad \text{sample well}} \right)}{{{OD}_{570}\quad {well}} + {medium}}} \right\rbrack$

following Espevik et al., J. Immunol. Meth. 95: 99-105 (1986). The results showed that even when the ratio of E⁺/E⁻ cells was as low as {fraction (1/45)}, significant production of TNF was observed, thus showing active CTLs. This led to the decision to test the drug resistant transfectants in groups of 30.

EXAMPLE 18

Cells were tested for TNF production as discussed in Example 17, supra. A total of 100 groups of E⁻ cells (4×10⁶ cells/group) were tested following transfection, and 7×10⁴ independent geneticin resistant transfectants were obtained, for an average of 700 per group. Only one group of transfected cells led to a microculture which caused anti-E antigen CTL clone 82/30 to produce TNF. Of 300 clones tested, 8 were positive. These clones were then tested for lysis by anti-E CTL, using the standard ⁵¹Cr release assay, and were found to be lysed as efficiently as the original E⁺ cell line. The transfectant E.T1, discussed herein, had the same lysis pattern as did MEL2.2 for CTLs against antigens B,C,D and F.

The fact that only one transfectant presented the antigen out of 70,000 geneticin resistance transfectants may at first seem very low, but it is not. The work described supra for P815 showed an average frequency of {fraction (1/13,000)}. Human DNA recipient MEL2.2 appears to integrate 5 times less DNA than P1.HTR.

EXAMPLE 19

Once transfectant E.T1 was found, analysis had to address several questions including whether an E⁺ contaminant of the cell population was the cause. The analysis of antigen presentation, described supra, shows that E.T1 is B⁻ and C⁻, Dust like the recipient cell MEL2.2. It was also found to be HPRT⁻, using standard selection procedures. All E⁺ cells used in the work described herein, however, were HPRT⁺.

It was also possible that an E⁺ revertant of MEL2.2 was the source for E.T1. To test this, the observation by Perucho et al., Cell 22: 309-317 (1980), that cotransfected sequences usually integrate together at a single location of recipient genome was employed. If antigen E in a transfectant results from cotransfection with pSVtkneoβ, then sequences should be linked and deletion of the antigen might also delete the neighboring pSVtkneoβ sequences. Wölfel et al., supra, has shown this to be true. If a normally E⁻ cell is transfected with pSVtkneoβ, then sequences should be linked and deletion of the antigen might also delete the neighboring pSVtkneoβ sequences. If a normally E⁺ cell transfected with pSVtkneoβ is E.T1, however, “co-deletion” should not take place. To test this, the transfectant E.T1 was subjected to immunoselection with 82/30, as described supra. Two antigen loss variants were obtained, which resisted lysis by this CTL. Neither of these had lost geneticin resistance; however, Southern blot analysis showed loss of several neo^(r) sequences in the variants, showing close linkage between the E gene and neo^(r) gene in E.T1, leading to the conclusion that E.T1 was a transfectant.

EXAMPLE 20

The E⁺ subclone MZ2-MEL 4B was used as a source of DNA for preparation of a cosmid library. This library of nearly 700,000 cosmids was transfected into MZ2-MEL 2.2 cells, following the cosmid transfection protocols described supra.

By packaging the DNA of cosmid transfectants directly into lambda phase components, it is sometimes possible to retrieve cosmids that contain the sequences of interest. This procedure was unsuccessful here, so we rescued the transfected sequence by ligating DNA of the transfectant to appropriate restriction fragments of cosmid vector pTL6. This was tried with two transfectants and was successful with one of them. One cosmid, referred to as B3, was recovered from this experiment, and subjected to restriction endonuclease digestion via XmaI, or by BamHI digestion of a large, 12 kb XmaI transfected fragment. The fragments were cloned into vector pTZ 18R, and then transfected into MEL2.2. Again, TNF production was the measure by which successful transfection was determined. The experiments led to the determination of a gene sequence capable of transfecting antigen E on the 12 kb XmaI fragment, and then on the 2.4 kb fragment of BamHI digestion of the 12 kb segment.

The 2.4 kb fragment hybridizes with a 2.4 kb fragment from MZ2-MEL and with a T cell clone of patient MZ-2, as determined by Southern Blots (BamHI/SmaI digested DNA). The band is absent from E⁻ antigen loss variants of MZ2-MEL, as seen in FIG. 12.

The sequence for the E antigen precursor gene has been determined, and is presented immediately below, and also in SEQ ID NO: 7.

    |   10     |   20     |   30     |   40     |   50     |   60 1 GGATCCAGGC CCTGCCAGGA AAAATATAAG GGCCCTGCGT GAGAACAGAG GGGGTCATCC 60 61 ACTGCATGAG AGTGGGGATG TCACAGAGTC CAGCCCACCC TCCTGGTAGC ACTGAGAAGC 120 121 CAGGGCTGTG CTTGCGGTCT GCACCCTGAG GGCCCGTGGA TTCCTCTTCC TGGAGCTCCA 180 181 GGAACCAGGC AGTGAGGCCT TGGTCTGAGA CAGTATCCTC AGGTCACAGA GCAGAGGATG 240 241 CACAGGGTGT GCCAGCAGTG AATGTTTGCC CTGAATGCAC ACCAAGGGCC CCACCTGCCA 300 301 CAGGACACAT AGGACTCCAC AGAGTCTGGC CTCACCTCCC TACTGTCAGT CCTGTAGAAT 360 361 CGACCTCTGC TGGCCGGCTG TACCCTGAGT ACCCTCTCAC TTCCTCCTTC AGGTTTTCAG 420 421 GGGACAGGCC AACCCAGAGG ACAGGATTCC CTGGAGGCCA CAGAGGAGCA CCAAGGAGAA 480 481 GATCTGTAAG TAGGCCTTTG TTAGAGTCTC CAAGGTTCAG TTCTCAGCTG AGGCCTCTCA 540 541 CACACTCCCT CTCTCCCCAG GCCTGTGGQT CTTCATTGCC CAGCTCCTGC CCACACTCCT 600 601 GCCTGCTGCC CTGACGAGAG TCATCATGTC TCTTGAGCAG AGGAGTCTGC ACTGCAAGCC 660 661 TGAGGAAGCC CTTGAGGCCC AACAAGAGGC CCTGGGCCTG GTGTGTGTGC AGGCTGCCAC 720 721 CTCCTCCTCC TCTCCTCTGG TCCTGGGCAC CCTGGAGGAG GTGCCCACTG CTGGGTCAAC 780 781 AGATCCTCCC CAGAGTCCTC AGGGAGCCTC CGCCTTTCCC ACTACCATCA ACTTCACTCG 840 841 ACAGAGGCAA CCCAGTGAGG CTTCCAGCAG CCGTGAAGAG GAGGGGCCAA GCACCTCTTG 900 901 TATCCTGGAG TCCTTGTTCC GAGCAGTAAT CACTAAGAAG GTGGCTGATT TGGTTGGTTT 960 961 TCTGCTCCTC AAATATCGAG CCAGGGAGCC AGTCACAAAG GCAGAAATGC TGGAGAGTGT 1020 1021 CATCAAAAAT TACAAGCACT GTTTTCCTGA GATCTTCGGC AAAGCCTCTG AGTCCTTGCA 1080 1081 GCTGGTCTTT GGCATTGACG TGAAGGAAGC AGACCCCACC GGCCACTCCT ATGTCCTTGT 1140 1141 CACCTGCCTA GGTCTCTCCT ATGATGGCCT GCTGGGTGAT AATCAGATCA TGCCCAAGAC 1200 1201 AGGCTTCCTG ATAATTGTCC TGGTCATGAT TGCAATGGAG GGCGGCCATG CTCCTGAGGA 1260 1261 GGAAATCTGG GAGGAGCTGA GTGTGATGGA GGTGTATGAT GGGAGGGAGC ACAGTGCCTA 1320 1321 TGGGGAGCCC AGGAAGCTGC TCACCCAAGA TTTGGTGCAG GAAAAGTACC TGGAGTACCG 1380 1381 GCAGGTGCCG GACAGTGATC CCGCACGCTA TGAGTTCCTG TGGGGTCCAA GGGCCCTCGC 1440 1441 TGAAACCAGC TATGTGAAAG TCCTTGAGTA TGTGATCAAG GTCAGTGCAA GAGTTCGCTT 1500 1501 TTTCTTCCCA TCCCTGCGTG AAGCAGCTTT GAGAGAGGAG GAAGAGGGAG TCTGAGCATG 1560 1561 AGTTGCAGCC AAGGCCAGTG GGAGGGGGAC TGGGCCAGTG CACCTTCCAG GGCCGCGTCC 1620 1621 AGCAGCTTCC CCTGCCTCGT GTGACATGAG GCCCATTCTT CACTCTGAAG AGAGCGGTCA 1680 1681 GTGTTCTCAG TAGTAGGTTT CTGTTCTATT GGGTGACTTG GAGATTTATC TTTGTTCTCT 1740 1741 TTTGGAATTG TTCAAATGTT TTTTTTTAAG GGATGGTTGA ATGAACTTCA GCATCCAAGT 1800 1801 TTATGAATGA CAGCAGTCAC ACAGTTCTGT GTATATAGTT TAAGGGTAAG AGTCTTGTGT 1960 1861 TTTATTCAGA TTGGGAAATC CATTCTATTT TGTGAATTGG GATAATAACA GCAGTGGAAT 1920 1921 AAGTACTTAG AAATGTGAAA AATGAGCAGT AAAATAGATG AGATAAAGAA CTAAAGAAAT 1980 1981 TAAGAGATAG TCAATTCTTG CCTTATACCT CAGTCTATTC TGTAAAATTT TTAAAGATAT 2040 2041 ATGCATACCT GGATTTCCTT GCCTTCTTTG AGAATGTAAG AGAAATTAAA TCTGAATAAA 2100 2101 GAATTCTTCC TGTTCACTGG CTCTTTTCTT CTCCATGCAC TGAGCATCTG CTTTTTGGAA 2160 2161 GGCCCTGGGT TAGTAGTGGA GATGCTAAGG TAAGCCAGAC TCATACCCAC CCATAGGGTC 2220 2221 GTAGAGTCTA GGAGCTGCAG TCACGTAATC GAGGTGGCAA GATGTCCTCT AAAGATGTAG 2280 2281 GGAAAAGTGA GAGAGGGGTG AGGGTGTGGG GCTCCGGGTG AGAGTGGTGG AGTGTGAATG 2340 2341 CCCTGAGCTG GGGCATTTTG GGCTTTGGGA AACTGCAGTT CCTTCTGGGG GAGCTGATTG 2400 2401 TAATGATCTT GGGTGGATCC 2420     |   10     |   20     |   30     |   40     |   50     |   60

EXAMPLE 21

After the 2.4 kb genomic segment had been identified, studies were carried out to determine if an “E⁺” subline expressed any homologous DNA. Cell line MZ2-MEL 3.0 was used as a source, and a cDNA library was prepared from its mRNA, using art known techniques. The 2.4 kb segment was used as a probe, and mRNA of about 1.8 kb was identified as homologous, using Northern blot analysis. When cDNA was screened, clones were obtained showing almost complete identity to parts of the 2.4 kb fragment. Two exons were thus identified. An additional exon was located upstream of these, via sequencing segments of cosmid B3 located in front of the 2.4 kb BamHI fragment. The gene extends over about 4.5 kb, as shown in FIG. 8. The starting point of the transcribed region was confirmed using PCR for the 5′ end of the cDNA. The three exons comprise 65, 73, and 1551 base pairs. An ATG is located at position 66 of exon 3, followed by an 828 base pair reading frame.

EXAMPLE 22

To determine if smaller segments of the 2.4 kb fragment could transfer the expression of antigen E, smaller pieces corresponding to the larger gene were prepared, using art recognized techniques, and transferred into E⁻ cells. FIG. 8 shows the boundaries of the three segments.

Transfer of antigen expression in this manner indicates that the gene codes for the antigen precursor, rather than coding for a protein which activates the antigen.

EXAMPLE 23

The probing of cDNA described supra revealed, surprisingly, two different but closely related cDNAs. These cDNAs, when tested, did not transfer expression of antigen E, but they do show substantial homology to the first cDNA segment. The three segments, appear to indicate a newly recognized family of genes, referred to as “MAGE” for “melanoma antigen”. In FIG. 9, “mage-1” directs expression of the antigen from MZ2 cells. Portions of the third exon of each gene are presented in FIG. 9. The second and third sequences are more closely related to each other than the first (18.1 and 18.9% difference compared to the first; 12% with each other). Out of 9 cDNA clones obtained, three of each type were obtained, suggesting equal expression. “MAGE” as used hereafter refers to a family of molecules, and the nucleic acids coding for them. These nucleic acids share a certain degree of homology and are expressed in tumor cells including several types of human tumor cells as well as in human tumors. The family is referred to as “MAGE” because the first members were identified in human melanoma cells. As the experiments which follow indicate, however, the members of the MAGE family are not at all restricted to melanoma tumors; rather, MAGE refers to a family of tumor rejection antigen precursors and the nucleic acid sequences coding therefore. The antigens resulting therefrom are referred to herein as “MAGE TRAs” or “melanoma antigen tumor rejection antigens”

EXAMPLE 24

Experiments with mouse tumors have demonstrated that new antigens recognized by T cells can result from point mutations that modify active genes in a region that codes for the new antigenic peptide. New antigens can also arise from the activation of genes that are not expressed in most normal cells. To clarify this issue for antigen MZ2-E, the mage-1 gene present in the melanoma cells was compared to that present in normal cells of patient MZ2. Amplification by polymerase chain reaction (PCR) of DNA of phytohemagglutinin-activated blood lymphocytes using primers surrounding a 1300 bp stretch covering the first half of the 2.4 kb fragment was carried out. As expected, a PCR product was obtained whereas none was obtained with the DNA of the E⁻ variant. The sequence of this PCR product proved identical to the corresponding sequence of the gene carried by the E⁺ melanoma cells. Moreover, it was found that antigen MZ2-E was expressed by cells transfected with the cloned PCR product. This result suggests that the activation of a gene normally silent is responsible for the appearance of tumor rejection antigen MZ2-E.

EXAMPLE 25

In order to evaluate the expression of gene mage-1 by various normal and tumor cells, Northern blots were hybridized with a probe covering most of the third exon. In contrast with the result observed with human tumor cell line MZ2-MEL 3.0, no band was observed with RNA isolated from a CTL clone of patient MZ2 and phytohemagglutinin-activated blood lymphocytes of the same patient. Also negative were several normal tissues of other individuals (FIG. 10 and FIG. 11). Fourteen melanoma cell lines of other patients were tested. Eleven were positive with bands of varying intensities. In addition to these culture cell lines, four samples of melanoma tumor tissue were analyzed. Two samples, including a metastasis of patient MZ2 proved positive, excluding the possibility that expression of the gene represented a tissue culture artefact. A few tumors of other histological types, including lung tumors were tested. Most of these tumors were positive (FIGS. 10 and 11). These results indicated that the MAGE gene family is expressed by many melanomas and also by other tumors. However, they provided no clear indication as to which of genes mage-1, 2 or 3 were expressed by these cells, because the DNA probes corresponding to the three genes cross-hybridized to a considerable extent. To render this analysis more specific, PCR amplification and hybridization with highly specific oligo- nucleotide probes were used. cDNAs were obtained and amplified by PCR using oligonucleotide primers corresponding to sequences of exon 3 that were identical for the three MAGE genes discussed herein. The PCR products were then tested for their ability to hybridize to three other oligonucleotides that showed complete specificity for one of the three genes (FIG. 9). Control experiments carried out by diluting RNA of melanoma MZ2-MEL 3.0 in RNA from negative cells indicated that under the conditions used herein the intensity of the signal decreased proportionally to the dilution and that positive signals could still be detected at a dilution of {fraction (1/300)}. The normal cells (lymphocytes) that were tested by PCR were confirmed to be negative for the expression of the three MAGE genes, suggesting therefore a level of expression of less than {fraction (1/300)}th that of the MZ2 melanoma cell line (FIG. 11). For the panel of melanoma cell lines, the results clearly showed that some melanomas expressed MAGE genes mage 1, 2 and 3 whereas other expressed only mage-2 and 3 (FIGS. 11 and 10). Some of the other tumors also expressed all three genes whereas others expressed only mage-2 and 3 or only mage-3. It is impossible to exclude formally that some positive PCR results do not reflect the expression of one of the three characterized MAGE genes but that of yet another closely related gene that would share the sequence of the priming and hybridizing oligonucleotides. It can be concluded that the MAGE gene family is expressed by a large array of different tumors and that these genes are silent in the normal cells tested to this point.

EXAMPLE 26

The availability of a sequence that transfects at high efficiency and efficiently expresses a TRAP made it possible to search for the associated major histocompatibility complex (MHC) class I molecule. The class I specificities of patient MZ2 are HLA-A1, A29, B37, B44 and C6. Four other melanomas of patients that had Al in common with MZ2 were cotransfected with the 2.4 kb fragment and pSVtkneoβ. Three of them yielded neo^(r) transfectants that stimulated TNF release by anti-E CTL clone 82/30, which is CD8+ (FIG. 10). No E- transfectant was obtained with four other melanomas, some of which shared A29, B44 or C6 with MZ2. This suggests that the presenting molecule for antigen MZ2-E is HLA-A1. In confirmation, it was found that, out of 6 melanoma cell lines derived from tumors of HLA-A1 patients, two stimulated TNF release by anti-E CTL clone 82/30 of patient MZ2. One of these tumor cell lines, MI13443-MEL also showed high sensitivity to lysis by these anti-E CTL. These two melanomas were those that expressed mage-1 gene (FIG. 13). Eight melanomas of patients with HLA haplotypes that did not include A1 were examined for their sensitivity to lysis and for their ability to stimulate TNF release by the CTL. None was found to be positive. The ability of some human anti-tumor CTL to lyse allogeneic tumors sharing an appropriate HLA specificity with the original tumor has been reported previously (Darrow, et al., J. Immunol. 142: 3329 (1989)). It is quite possible that antigenic peptides encoded by genes mage 2 and 3 can also be presented to autologous CTL by HLA-A1 or other class I molecules, especially in view of the similar results found with murine tumors, as elaborated upon supra.

EXAMPLE 27

As indicated supra, melanoma MZ2 expressed antigens F, D and A′, in addition to antigen E. Following the isolation of the nucleic acid sequence coding for antigen E, similar experiments were carried out to isolate the nucleic acid sequence coding for antigen F.

To do this, cultures of cell line MZ2-MEL2.2, an E⁻ cell line described supra, were treated with anti-P CTL clone 76/6, in the same manner described for treatment with anti-E CTL clones. This resulted in the isolation of an F antigen loss variant, which was then subjected to several rounds of selection. The resulting cell line, “MZ2-MEL2.2.5” was completely resistant to lysis by anti-F CTLs, yet proved to be lysed by anti-D CTLs.

Again, following the protocols set forth for isolation of antigen -E precursor DNA, the F⁻ variant was transfected with genomic DNA from F⁺ cell line MZ2-MEL3.0. The experiments yielded 90,000 drug resistant transfectants. These were tested for MZ2-F expression by using pools of 30 cells in the TNF detection assay elaborated upon supra. One pool stimulated TNF release by anti-F CTLs, and was cloned. Five of 145 clones were found to stimulate anti-F CTLs. Lysis assays, also following protocols described supra, confirmed (i) expression of the gene coding for antigen F, and (ii) presentation of antigen F itself.

EXAMPLE 28

Following identification of F⁺ cell lines, the DNA therefrom was used to transfect cells. To do this, a cosmid library of F⁺ cell line MZ2-MEL.43 was prepared, again using the protocols described supra. The library was divided into 14 groups of about 50,000 cosmids, and DNA from each group was transfected into MZ2-MEL2.2.5. Transfectants were then tested for their ability to stimulate TNF release from anti-F CTL clone 76/6. Of 14 groups of cosmids, one produced two independent transfectants expressing antigen F; a yield of two positives out of 17,500 geniticin resistant transfectants.

EXAMPLE 29

The existence of a gene family was suggested by the pattern observed on the Southern blot (FIG. 12). To do this, the 2.4 kb BamHI fragment, which transferred the expression of antigen M22-E, was labelled with 32p and used as a probe on a Southern Blot of BamHI digested DNA of E+cloned subclone M22-MEL2.2. Hybridization conditions included 50 μl/cm² of 3.5×SSC, 1×Denhardt's solution; 25 mM sodium phosphate buffer (pH 7.0), 0.5% SDS, 2 mM EDTA, where the 2.4 kb probes had been labelled with [α³²p]dCTP (2-3000 Ci/mole), at 3×10⁶ cpm/ml. Hybridization was carried out for 18 hours at 65° C. After this, the membranes were washed at 65° C. four times for one hour each in 2×SSC, 0.1% SDS, and finally for 30 minutes in 0.1×SSC, 0.1% SDS. To identify hybridization, membranes were autoradiographed using Kodak X-AR film and Kodak X-Omatic fine intensifying screens.

In the following examples, whenever “hybridization” is referred to, the stringency conditions used were similar to those described supra. “Stringent conditions” as used herein thus refers to the foregoing conditions; subject to routine, art recognized modification.

EXAMPLE 30

The cDNA coding for mage 4 was identified from a sample of the human sarcoma cell line LB23-SAR. This cell line was found to not express mage 1, 2 or 3, but the mRNA of the cell line did hybridize to the 2.4 kb sequence for mage 1. To study this further, a cDNA library was prepared from total LB23-SAR mRNA, and was then hybridized to the 2.4 kb fragment. A cDNA sequence was identified as hybridizing to this probe, and is identified hereafter as mage 4.

EXAMPLE 31

Experiments were carried out using PHA-activated lymphocytes from patient “MZ2”, the source of the “MZ” cells discussed supra. An oligonucleotide probe which showed homology to mage 1 but not mage 2 or 3 was hybridized with a cosmid library derived from the PHA activated cells. The size of the hybridizing BamHI cosmid fragment, however, was 4.5 kb, thus indicating that the material was not mage 1; however, on the basis of homology to mage 1-4, the fragment can be referred to as “mage 5”. The sequence of MAGE 5 is presented in SEQ ID NO: 16.

EXAMPLE 32

Melanoma cell line LB-33-MEL was tested. Total mRNA from the cell line was used to prepare cDNA, which was then amplified with oligos CHO9: (ACTCAGCTCCTCCCAGATTT), and CHO10: (GAAGAGGAGGGGCCAAG). These oligos correspond to regions of exon 3 that are common to previously described mage 1, 2 and 3.

To do this, 1 μg of RNA was diluted to a total volume of 20 μl, using 2 μl of 10×PCR buffer, 2 μl of each of 10 mM dNTP, 1.2 μl of 25 mM MgCl₂, 1 μl of an 80 mM solution of CHO9, described supra, 20 units of RNAsin, and 200 units of MMLV reverse transcriptase. This was followed by incubation for 40 minutes at 42° C. PCR amplification followed, using 8 μl of 10×PCR buffer, 4.8 μl of 25 mM MgCl₂, 1 μl of CHO10, 2.5 units of Thermus acquaticus (“Taq”) polymerase, and water to a total volume of 100 μl. Amplification was then carried out for 30 cycles (1 minute 94° C.; 2 minutes at 52° C., 3 minutes at 72° C.). Ten μl of each reaction were then size fractionated on agarose gel, followed by nitrocellulose blotting. The product was found to hybridize with oligonucleotide probe CHO18 (TCTTGTATCCTGGAGTCC). This probe identified mage 1 but not mage 2 or 3. However, the product did not hybridize to probe SEQ 4 (TTGCCAAGATCTCAGGAA). This probe also binds mage 1 but not 2 and 3. This indicated that the PCR product contained a sequence that differed from mage 1, 2 and 3. Sequencing of this fragment also indicated differences with respect to mage 4 and 5. These results indicate a sequence differing from previously identified mage 1, 2, 3, 4 and 5, and is named mage 6.

EXAMPLE 33

In additional experiments using cosmid libraries from PHA-activated lymphocytes of MZ2, the 2.4 kb mage 1 fragment was used as a probe and isolated a complementary fragment. This clone, however, did not bind to oligonucleotides specific for mage 1, 2, 3 or 4. The sequence obtained shows some homology to exon 3 of mage 1, and differs from mages 1-6. It is referred to as mage 7 hereafter. Additional screenings yielded mage 8-11.

EXAMPLE 34

The usefulness of the TRAPs, as well as TRAs derived therefrom, was exemplified by the following. Exon 3 of mage 1 was shown to transfer expression of antigen E. As a result, it was decided to test whether synthetic peptides derived from this exon 3 could be used to confer sensitivity to anti-E CTL.

To do this, and using standard protocols, cells normally insensitive to anti-E/CTLs were incubated with the synthetic peptides derived from Exon 3.1. Using the CTL lytic assays described supra on P815A, and a peptide concentration of 3 mM, the peptide Glu-Ala-Asp-Pro-Thr-Gly-His-Ser-Tyr was shown to be best. The assay showed lysis of 30%, indicating conferring of sensitivity to the anti-E CTL.

EXAMPLE 35

Nucleic acid sequences referred to as “smage” were isolated from murine cells. Using the protocols described supra, a cosmid library was prepared from the DNA of normal DBA/2 kidney cells, using cosmid vector C2RB. As a probe, the 2.4 kb BamHI fragment of MAGE-1 was used. The DNA was blotted to nylon filters, and these were washed in 2×SSC at 65° C. to identify the smage material.

EXAMPLE 36

Further tissue samples were tested for the presence of MAGE genes, using the protocols discussed supra. Some of these results follow.

There was no expression of the MAGE genes in brain or kidney tumor tissue. Colon tumor tissue showed expression of MAGE 1, 2, 3 and 4, although not all tumors tested showed expression of all MAGE genes. This is also true for pancreatic tumor (MAGE 1); non-small cell lung (MAGE 1, 2, 3 and 4), prostate (MAGE 1), sarcomas (MAGE 1, 2, 3 and 4), breast (MAGE 1, 2 and 3), and larynx (MAGE 1 and 4).

EXAMPLE 37

A cytolytic CTL clone “20/38” was obtained from peripheral blood lymphocytes of melanoma patient MZ2. This clone is described by Van den Eynde et al., Int. J. Cancer 44: 634-640 (1989), the disclosure of which is incorporated by reference. The CTL clone has isolated following Herin et al., Int. J. Cancer 39: 390-396 (1987), which is incorporated by reference. The assay is described herein, however. Autologous melanoma cells were grown in vitro, and then resuspended at 10⁷ cells/ml in DMEM, supplemented with 10% HEPES and 30% FCS, and incubated for 45 minutes at 37° C. with 200 μCi/ml of Na(⁵¹Cr)O₄. Labelled cells were washed three times with DMEM, supplemented with 10 mM HEPES. These were then resuspended in DMEM supplemented with 10 mM HEPES and 10% FCS, after which 100 μl aliquots containing 10³ cells, were distributed into 96 well microplates. Samples of the CTL clone were added in 100 ul of the same medium, and assays were carried out in duplicate. Plates were centrifuged for four minutes at 100 g, and incubated for four hours at 37° C. in a 5.5% CO₂ atmosphere.

Plates were centrifuged again, and 100 ul aliquots of supernatant were collected and counted. Percentage of ⁵¹Cr release was calculated as follows: ${\% \quad {\,^{51}{Cr}}\quad {release}} = {\frac{\left( {{ER} - {SR}} \right)}{\left( {{MR} - {SR}} \right)} \times 100}$

where ER is observed, experimental ⁵¹Cr release, SR is spontaneous release measured by incubating 10³ labeled cells in 200 ul of medium alone, and MR is maximum release, obtained by adding 100 ul 0.3% Triton X-100 to target cells.

Those mononuclear blood samples which showed high CTL activity were expanded and cloned via limiting dilution, and were screened again, using the same methodology.

The same method was used to test target K562 cells. When EBV-B cells were used, the only change was the replacement of DMEM medium by Hank's medium, supplemented with 5% FCS.

These experiments led to isolation of CTL clone 20/38.

FIG. 1 presents the results of these assays. Specifically, it will be seen that the CTL clone lysed autologous melanoma cell line MZ2-MEL.3.0, but did not lyse EBV-B cell lines, fibroblasts, K562 or non-autologous melanoma cell line SK-MEL-29.

EXAMPLE 38

Once the CTL clone was recognized as being specific for the autologous cell line, it was tested for antigenic specificity. To do this, antigen loss variants derived from patient MZ2 were tested in the same type of chromium release assay described above. These target lines were MZ2-MEL 3.0, which is D⁺, E⁺, F⁺, A⁺, MZ2-MEL.61, which is D⁻, MZ2-MEL 2.2, which is E⁻, and MZ2-MEL.4, which is F⁻. In addition to CTL clone 20/38, clones which are known to be anti-A (CTL 28/336), anti-F (CTL 76/6), and anti-E (CTL 22/13) were tested.

These results are set forth in FIG. 15. It will be seen that CTL clone 20/38 lysed all the cell lines leading to chromium release except D⁻ cell line MZ2-MEL.61, thus indicating that the CTL clone is anti-D. This result was confirmed, in experiments not included herein, by experiments where TNF release by the CTL clone was observed only in the presence of melanoma lines presenting antigen D.

EXAMPLE 39

Once antigen D was identified as the target molecule, studies were carried out to determine the HLA type which presented it. The experiments described in example A showed that antigen D was presented by MZ2-MEL, and this cell line's HLA specificity is known (i.e., A1, A29, B37, B44, Cw6, C.cl.10). It was also known, however, that a variant of MZ2-MEL which had lost HLA molecules A29, B44 and C.cl.10 still expressed antigen D, so these could be eliminated from consideration. Studies were not carried out on lines expressing B37, as none could be found.

In all, 13 allogeneic lines were tested, which expressed either HLA-A1 (10 of 13), or Cw6 (3 of 13). The cell lines were tested for their ability to stimulate release of TNF by CTL clone 20/38, using the method of Traversari et al., Immunogenetics 35: 145-152 (1992), the disclosure of which is incorporated by reference. This assay measures TNF release via testing toxicity of supernatants on WEHI 164-13 cells.

In the assays, cell samples (3000, 10,000 or 30,000 cells) from the allogeneic lines were cultured in the presence of 1500 cells of the CTL clone, and 25 u/ml of IL-2. Twenty-four hours later, the supernatant from the culture was tested against the WEHI cells for toxicity. The results are presented in Table 3, which follows.

Eight cell lines were found to stimulate TNF release from the CTL clone 20/38. All of these lines were HLA-A1. None of the Cw6 presenting lines did so.

The cell lines were also assayed to determine MAGE expression. All eight of the lines which stimulated TNF release expressed MAGE-3, whereas the two HLA-A1 lines which were negative did not.

EXAMPLE 40

In view of the results set forth in example C, experiments were carried out to determine if antigen D was in fact a tumor rejection antigen derived from MAGE-3. To do this, recipient COS7 cells were transfected with 100 ng of the gene for HLA-A1 cloned into pcDNA I/Amp, and 100 ng of one of (a) cDNA for MAGE-1 cloned into pcDNA I/Amp, (b) cDNA for MAGE-2 cloned into pcDSRα, or (c) cDNA for MAGE-3 cloned into pcDSRα. The transfecting sequences were ligated into the plasmids in accordance with manufacturer's instructions. Samples of COS-7 cells were seeded, at 15,000 cells/well into tissue culture flat bottom microwells, in Dulbeco's modified Eagles Medium (“DMEM”) supplemented with 10% fetal calf serum. The cells were incubated overnight at 37° C., medium was removed and then replaced by 30 μl/well of DMEM medium containing 10% Nu serum, 400 μg/ml DEAE-dextran, 100 μM chloroquine, and the plasmids described above. Following four hours of incubation at 37° C., the medium was removed, and replaced by 50 μl of PBS containing 10% DMSO. This medium was removed after two minutes and replaced by 200 μl of DMEM supplemented with 10% of FCS.

Following this change in medium, COS cells were incubated for 24 hours at 37° C. Medium was then discarded, and 1500 cells of CTL clones 20/38 were added, in 100 μl of Iscove medium containing 10% pooled human serum, supplemented with 25 u/ml of IL-2. Supernatant was removed after 24 hours, and TNF content was determined in an assay on WEHI cells, as described by Traversari et al., Immunogenetics 35: 145-152 (1992), the disclosure of which is incorporated by reference. These results are shown in FIG. 16.

It will be seen that the CTL clone was strongly stimulated by COS7 cells transfected with HLA-A1 and MAGE-3, but not by the cells transfected with the other mage genes. This leads to the conclusion that antigen D is a tumor rejection antigen derived from the tumor rejection antigen precursor coded by gene MAGE-3, and that this TRA is presented by HLA-A1 molecules.

The foregoing disclosure, including the examples, places many tools of extreme value in the hands of the skilled artisan. To begin, the examples identify and provide a methodology for isolating nucleic acid molecules which code for tumor rejection antigen precursors as well as the nucleic acid molecules complementary thereto. It is known that DNA exists in double stranded form, and that each of the two strands is complementary to the other. Nucleic acid hybridization technology has developed to the point where, given a strand of DNA, the skilled artisan can isolate its complement, or synthesize it.

“Nucleic acid molecule” as used herein refers to all species of DNA and RNA which possess the properties discussed supra. Genomic and complementary DNA, or “cDNA” both code for particular proteins, and as the examples directed to isolation of MAGE coding sequences show, this disclosure teaches the artisan how to secure both of these.

Similarly, RNA molecules, such as mRNA can be secured. Again, with reference to the skilled artisan, once one has a coding sequence in hand, mRNA can be isolated or synthesized.

Complementary sequences which do not code for TRAP, such as “antisense DNA” or mRNA are useful, e.g., in probing for the coding sequence as well as in methodologies for blocking its expression.

It will also be clear that the examples show the manufacture of biologically pure cultures of cell lines which have been transfected with nucleic acid sequences which code for or express the TRAP molecules. Such cultures can be used as a source for tumor rejection antigens, e.g., or as therapeutics. This aspect of the invention is discussed infra.

Cells transfected with the TRAP coding sequences may also be transfected with other coding sequences. Examples of other coding sequences include cytokine genes, such as interleukins (e.g., IL-2 or IL-4), or major histocompatibility complex (MHC) or human leukocyte antigen (HLA) molecules. Cytokine gene transfection is of value because expression of these is expected to enhance the therapeutic efficacy of the biologically pure culture of the cells in vivo. The art is well aware of therapies where interleukin transfectants have been administered to subjects for treating cancerous conditions. In a particularly preferred embodiment, cells are transfected with sequences coding for each of (i) a TRAP molecule, (ii) an HLA/MHC molecule, and (iii) a cytokine.

Transfection with an MHC/HLA coding sequence is desirable because certain of the TRAs may be preferentially or specifically presented only by particular MHC/HLA molecules. Thus, where a recipient cell already expresses the MHC/HLA molecule associated with presentation of a TRA, additional transfection may not be necessary although further transformation could be used to cause over-expression of the antigen. On the other hand, it may be desirable to transfect with a second sequence when the recipient cell does not normally express the relevant MHC/HLA molecule. It is to be understood, of course, that transfection with one additional sequence does not preclude further transfection with other sequences.

The term “biologically pure” as used in connection with the cell line described herein simply means that these are essentially free of other cells. Strictly speaking, a “cell line” by definition is “biologically pure”, but the recitation will establish this fully.

Transfection of cells requires that an appropriate vector be used. Thus, the invention encompasses expression vectors where a coding sequence for the TRAP of interest is operably linked to a promoter. The promoter may be a strong promoter, such as those well known to the art, or a differential promoter, i.e., one which is operative only in specific cell types. The expression vectors may also contain all or a part of a viral or bacterial genome, such as vaccinia virus or BCG. Such vectors are especially useful in preparing vaccines.

The expression vectors may incorporate several coding sequences, as long as the TRAP sequence is contained therein. The cytokine and/or MHC/HLA genes discussed supra may be included in a single vector with the TRAP sequence. Where this is not desired, then an expression system may be provided, where two or more separate vectors are used where each coding sequence is operably linked to a promoter. Again, the promoter may be a strong or differential promoter. Co-transfection is a well known technique, and the artisan in this field is expected to have this technology available for utilization. The vectors may be constructed so that they code for the TRA molecule directly, rather than the TRAP molecule. This eliminates the need for post-translational processing.

As the foregoing discussion makes clear, the sequences code for “tumor rejection antigen precursors” (“TRAPs”) which, in turn, are processed into tumor rejection antigens (“TRAs”) Isolated forms of both of these categories are described herein, including specific examples of each. Perhaps their most noteworthy aspect is as vaccines for treating various cancerous conditions. The evidence points to presentation of TRAs on tumor cells, followed by the development of an immune response and deletion of the cells. The examples show that when various TRAs are administered to cells, a CTL response is mounted and presenting cells are deleted. This is behavior characteristic of vaccines, and hence TRAPs, which are processed into TRAs, and the TRAs themselves may be used, either alone or in pharmaceutically appropriate compositions, as vaccines. Similarly, presenting cells may be used in the same manner, either alone or as combined with ingredients to yield pharmaceutical compositions. Additional materials which may be used as vaccines include isolated cells which present the TRA molecule on their surface, as well as TRAP fragments, mutated viruses, especially etiolated forms, and transfected bacteria. “Fragments” used herein refers to peptides which are smaller than the TRAP, but which possess the properties required of a vaccine, as discussed supra. Another vaccine comprises or consists of complexes of TRA and HLA molecule. Vaccines of the type described herein may be used preventively, i.e., via administration to a subject in an amount sufficient to prevent onset of a cancerous condition.

The generation of an immune response, be it T-cell or B-cell related, is characteristic of the effect of the presented tumor rejection antigen. With respect to the B-cell response, this involves, inter alia, the generation of antibodies to the TRA, i.e., which specifically bind thereto. In addition, the TRAP molecules are of sufficient size to render them immunogenic, and antibodies which specifically bind thereto are a part of this invention. These antibodies may be polyclonal or monoclonal, the latter being prepared by any of the well recognized methodologies for their preparation which need not be repeated here. For example, mAbs may be prepared using an animal model, e.g., a Balb/C mouse or in a test tube, using, e.g., EBV transformants. In addition, antiserum may be isolated from a subject afflicted with a cancerous condition where certain cells present a TRA. Such antibodies may also be generated to epitopes defined by the interaction of TRA and HLA/MHC molecules.

Review of the foregoing disclosure will show that there are a number of facets to the system which may be referred to as “tumor rejection antigen presentation and recognition”. Recognition of these phenomena has diagnostic consequences. For example, the existence of specific CTL clones, or antibodies to the TRA makes it possible to diagnose or monitor cancerous conditions (explained infra), by monitoring the CTLs in a sample from a subject, binding of antibodies to TRAs, or the activity of anti-TRA CTLs in connection with subject samples. Similarly, the expression of nucleic acid molecules for TRAPs can be monitored via amplification (e.g., “polymerase chain reaction”), anti-sense hybridization, probe technologies, and so forth. Various subject samples, including body fluids (blood, serum, and other exudates, e.g.), tissues and tumors may be so assayed.

A particular manner of diagnosis is to use an adaptation of the standard “tuberculin test” currently used for diagnosis of tuberculosis. This standard skin test administers a stable form of “purified protein derivative” or “PPD” as a diagnostic aid. In a parallel fashion, TRAs in accordance with this invention may be used in such a skin test as a diagnostic aid or monitoring method.

The term “cancerous condition” is used herein to embrace all physiological events that commence with the initiation of the cancer and result in final clinical manifestation. Tumors do not spring up “ab initio ” as visible tumors; rather there are various events associated with the transformation of a normal cell to malignancy, followed by development of a growth of biomass, such as a tumor, metastasis, etc. In addition, remission may be conceived of as part of “a cancerous condition” as tumors seldom spontaneously disappear. The diagnostic aspects of this invention include all events involved in carcinogenesis, from the first transformation to malignancy of a single cell, through tumor development and metastasis, as well as remission. All are embraced herein.

Where “subject” is used, the term embraces any species which can be afflicted with a cancerous condition. This includes humans and non-humans, such as domesticated animals, breeding stock, and so forth.

There are therapeutic aspects of this invention as well. The efficacy of administration of effective amounts of TRAPs and TRAs as vaccines has already been discussed supra. Similarly, one may develop the specific CTLs in vitro and then administer these to the subject. Antibodies may be administered, either polyclonal or monoclonal, which specifically bind to cells presenting the TRA of interest. These antibodies may be coupled to specific antitumor agents, including, but not being limited to, methotrexate radio-iodinated compounds, toxins such as ricin, other cytostatic or cytolytic drugs, and so forth. Thus, “targeted” antibody therapy is included herein, as is the application of deletion of the cancerous cells by the use of CTLs.

The data from examples 37-40 show that a tumor rejection antigen derived from MAGE-3 is presented by HLA-A1 molecules. As such, in addition to the nucleic acid molecules coding for this TRAP, the TRAP itself as coded for by the sequences, vectors, cell lines, etcetera which incorporate this nucleic acid molecule, the invention also encompasses combination of the molecules coding for the MAGE-3 TRAP and HLA-A1. Thus, co-transfectants, vectors containing coding sequences for both, expression systems such as kits, or separate vectors, and so forth, are all embraced by the invention. Similarly, the vaccines discussed supra can be made by incorporating the TRAP from MAGE-3 and an adjuvant.

It is to be understood that a given TRAP may yield more than one TRA. In the case of MAGE-3, it has been shown that antigen D, as the term is used herein, derives therefrom, and one aspect of the invention is this isolated tumor rejection antigen. Another is isolated complexes of the TRA and its presenting molecule, i.e., HLA-A1.

The identification of MAGE-3 derived TRAs as being presented by HLA-Al molecules suggests various therapeutic and diagnostic approaches. In a therapeutic context, e.g., the treatment of a disorder characterized by MAGE-3 expression may be treated in a number of ways, “disorder” being used to refer to any pathological condition where MAGE-3 TRAP is expressed, such as cancer (e.g., melanoma).

Therapeutic approaches based upon the disclosure are premised on a response by a subject's immune system, leading to lysis of TRA presenting cells, such as HLA-A1 cells. One such approach is the administration of CTLs specific to the complex to a subject with abnormal cells of the phenotype at issue. It is within the skill of the artisan to develop such CTLs in vitro. Specifically, a sample of cells, such as blood cells, are contacted to a cell presenting the complex and capable of provoking a specific CTL to proliferate. The target cell can be a transfectant, such as a COS cell of the type described supra. These transfectants present the desired complex on their surface and, when combined with a CTL of interest, stimulate its proliferation. COS cells, such as those used herein are widely available, as are other suitable host cells.

To detail the therapeutic methodology, referred to as adoptive transfer (Greenberg, J. Immunol. 136 (5): 1917 (1986); Reddel et al., Science 257: 238 (Jul. 10, 1992); Lynch et al., Eur. J. Immunol. 21: 1403-1410 (1991); Kast et al., Cell 59: 603-614 (Nov. 17, 1989)), cells presenting the desired complex are combined with CTLs leading to proliferation of the CTLs specific thereto. The proliferated CTLs are then administered to a subject with a cellular abnormality which is characterized by certain of the abnormal cells presenting the particular complex. The CTLs then lyse the abnormal cells, thereby achieving the desired therapeutic goal.

The foregoing therapy assumes that at least some of the subject's abnormal cells present the HLA/TRA complex. This can be determined very easily, as the art is very familiar with methods for identifying cells which present a particular HLA molecule, as well as how to identify cells expressing DNA containing the indicated sequences. Once isolated, such cells can be used with a sample of a subject's abnormal cells to determine lysis in vitro. If lysis is observed, then the use of specific CTLs in such a therapy may alleviate the condition associated with the abnormal cells. A less involved methodology examines the abnormal cells for HLA phenotyping, using standard assays, and determines expression via amplification using, e.g., PCR.

Adoptive transfer is not the only form of therapy that is available in accordance with the invention. CTLs can also be provoked in vivo, using a number of approaches. One approach, i.e., the use of non-proliferative cells expressing the complex, has been elaborated upon supra. The cells used in this approach may be those that normally express the complex, such as irradiated melanoma cells or cells transfected with one or both of the genes necessary for presentation of the complex. Chen et al., Proc. Natl. Acad. Sci. USA 88: 110-114 (January, 1991) exemplifies this approach, showing the use of transfected cells expressing HPVE7 peptides in a therapeutic regime. Various cell types may be used. Similarly, vectors carrying one or both of the genes of interest may be used. Viral or bacterial vectors are especially preferred. In these systems, the gene of interest is carried by, e.g., a Vaccinia virus or the bacteria BCG, and the materials de facto “infect” host cells. The cells which result present the complex of interest, and are recognized by autologous CTLs, which then proliferate. A similar effect can be achieved by combining the tumor rejection antigen or the precursor itself with an adjuvant to facilitate incorporation into HLA-A1 presenting cells which present the HLA molecule of interest. The TRAP is processed to yield the peptide partner of the HLA molecule while the TRA is presented without the need for further processing. Thus, one may treat disorders where a MAGE-3 derived TRA is presented by HLA-A1 molecules, or by any HLA molecule.

In a diagnostic context, one may determine a disorder, as the term is used herein, by assaying for expression of the TRAP. This can be done directly (via, e.g., a PCR assay for TRAP sequences), or indirectly, via assaying for a MAGE-3 derived TRA, as the TRA's presence means that the TRAP is or was expressed.

It will be noted that two nucleic acid molecules are presented herein, i.e., MAGE-3 and MAGE-31, each of which code for TRAP MAGE-3. It is to be understood that when the epxression “nucleic acid molecule which codes for MAGE-3 TRAP” is used, all molecules are covered which yield this molecule upon expression. Any number of variations, such as those showing codon degeneracy within the coding region, or variation within the introns, are covered by the invention.

The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, it being recognized that various modifications are possible within the scope of the invention.

30 462 base pairs nucleic acid single linear genomic DNA 1 ACCACAGGAG AATGAAAAGA ACCCGGGACT CCCAAAGACG CTAGATGTGT GAAGATCCTG 60 ATCACTCATT GGGTGTCTGA GTTCTGCGAT ATTCATCCCT CAGCCAATGA GCTTACTGTT 120 CTCGTGGGGG GTTTGTGAGC CTTGGGTAGG AAGTTTTGCA AGTTCCGCCT ACAGCTCTAG 180 CTTGTGAATT TGTACCCTTT CACGTAAAAA AGTAGTCCAG AGTTTACTAC ACCCTCCCTC 240 CCCCCTCCCA CCTCGTGCTG TGCTGAGTTT AGAAGTCTTC CTTATAGAAG TCTTCCGTAT 300 AGAACTCTTC CGGAGGAAGG AGGGAGGACC CCCCCCCTTT GCTCTCCCAG CATGCATTGT 360 GTCAACGCCA TTGCACTGAG CTGGTCGAAG AAGTAAGCCG CTAGCTTGCG ACTCTACTCT 420 TATCTTAACT TAGCTCGGCT TCCTGCTGGT ACCCTTTGTG CC 462 675 base pairs nucleic acid single linear genomic DNA 2 ATG TCT GAT AAC AAG AAA CCA GAC AAA GCC CAC AGT GGC TCA GGT GGT 48 Met Ser Asp Asn Lys Lys Pro Asp Lys Ala His Ser Gly Ser Gly Gly 5 10 15 GAC GGT GAT GGG AAT AGG TGC AAT TTA TTG CAC CGG TAC TCC CTG GAA 96 Asp Gly Asp Gly Asn Arg Cys Asn Leu Leu His Arg Tyr Ser Leu Glu 20 25 30 GAA ATT CTG CCT TAT CTA GGG TGG CTG GTC TTC GCT GTT GTC ACA ACA 144 Glu Ile Leu Pro Tyr Leu Gly Trp Leu Val Phe Ala Val Val Thr Thr 35 40 45 AGT TTT CTG GCG CTC CAG ATG TTC ATA GAC GCC CTT TAT GAG GAG CAG 192 Ser Phe Leu Ala Leu Gln Met Phe Ile Asp Ala Leu Tyr Glu Glu Gln 50 55 60 TAT GAA AGG GAT GTG GCC TGG ATA GCC AGG CAA AGC AAG CGC ATG TCC 240 Tyr Glu Arg Asp Val Ala Trp Ile Ala Arg Gln Ser Lys Arg Met Ser 65 70 75 80 TCT GTC GAT GAG GAT GAA GAC GAT GAG GAT GAT GAG GAT GAC TAC TAC 288 Ser Val Asp Glu Asp Glu Asp Asp Glu Asp Asp Glu Asp Asp Tyr Tyr 85 90 95 GAC GAC GAG GAC GAC GAC GAC GAT GCC TTC TAT GAT GAT GAG GAT GAT 336 Asp Asp Glu Asp Asp Asp Asp Asp Ala Phe Tyr Asp Asp Glu Asp Asp 100 105 110 GAG GAA GAA GAA TTG GAG AAC CTG ATG GAT GAT GAA TCA GAA GAT GAG 384 Glu Glu Glu Glu Leu Glu Asn Leu Met Asp Asp Glu Ser Glu Asp Glu 115 120 125 GCC GAA GAA GAG ATG AGC GTG GAA ATG GGT GCC GGA GCT GAG GAA ATG 432 Ala Glu Glu Glu Met Ser Val Glu Met Gly Ala Gly Ala Glu Glu Met 130 135 140 GGT GCT GGC GCT AAC TGT GCC TGT GTT CCT GGC CAT CAT TTA AGG AAG 480 Gly Ala Gly Ala Asn Cys Ala Cys Val Pro Gly His His Leu Arg Lys 145 150 155 160 AAT GAA GTG AAG TGT AGG ATG ATT TAT TTC TTC CAC GAC CCT AAT TTC 528 Asn Glu Val Lys Cys Arg Met Ile Tyr Phe Phe His Asp Pro Asn Phe 165 170 175 CTG GTG TCT ATA CCA GTG AAC CCT AAG GAA CAA ATG GAG TGT AGG TGT 576 Leu Val Ser Ile Pro Val Asn Pro Lys Glu Gln Met Glu Cys Arg Cys 180 185 190 GAA AAT GCT GAT GAA GAG GTT GCA ATG GAA GAG GAA GAA GAA GAA GAG 624 Glu Asn Ala Asp Glu Glu Val Ala Met Glu Glu Glu Glu Glu Glu Glu 195 200 210 GAG GAG GAG GAG GAA GAG GAA ATG GGA AAC CCG GAT GGC TTC TCA CCT 672 Glu Glu Glu Glu Glu Glu Glu Met Gly Asn Pro Asp Gly Phe Ser Pro 220 225 230 235 TAG 675 228 base pairs nucleic acid single linear genomic DNA 3 GCATGCAGTT GCAAAGCCCA GAAGAAAGAA ATGGACAGCG GAAGAAGTGG TTGTTTTTTT 60 TTCCCCTTCA TTAATTTTCT AGTTTTTAGT AATCCAGAAA ATTTGATTTT GTTCTAAAGT 120 TCATTATGCA AAGATGTCAC CAACAGACTT CTGACTGCAT GGTGAACTTT CATATGATAC 180 ATAGGATTAC ACTTGTACCT GTTAAAAATA AAAGTTTGAC TTGCATAC 228 1365 base pairs nucleic acid single linear genomic DNA 4 ACCACAGGAG AATGAAAAGA ACCCGGGACT CCCAAAGACG CTAGATGTGT 50 GAAGATCCTG ATCACTCATT GGGTGTCTGA GTTCTGCGAT ATTCATCCCT 100 CAGCCAATGA GCTTACTGTT CTCGTGGGGG GTTTGTGAGC CTTGGGTAGG 150 AAGTTTTGCA AGTTCCGCCT ACAGCTCTAG CTTGTGAATT TGTACCCTTT 200 CACGTAAAAA AGTAGTCCAG AGTTTACTAC ACCCTCCCTC CCCCCTCCCA 250 CCTCGTGCTG TGCTGAGTTT AGAAGTCTTC CTTATAGAAG TCTTCCGTAT 300 AGAACTCTTC CGGAGGAAGG AGGGAGGACC CCCCCCCTTT GCTCTCCCAG 350 CATGCATTGT GTCAACGCCA TTGCACTGAG CTGGTCGAAG AAGTAAGCCG 400 CTAGCTTGCG ACTCTACTCT TATCTTAACT TAGCTCGGCT TCCTGCTGGT 450 ACCCTTTGTG CC 462 ATG TCT GAT AAC AAG AAA CCA GAC AAA GCC CAC AGT GGC TCA 504 GGT GGT GAC GGT GAT GGG AAT AGG TGC AAT TTA TTG CAC CGG 546 TAC TCC CTG GAA GAA ATT CTG CCT TAT CTA GGG TGG CTG GTC 588 TTC GCT GTT GTC ACA ACA AGT TTT CTG GCG CTC CAG ATG TTC 630 ATA GAC GCC CTT TAT GAG GAG CAG TAT GAA AGG GAT GTG GCC 672 TGG ATA GCC AGG CAA AGC AAG CGC ATG TCC TCT GTC GAT GAG 714 GAT GAA GAC GAT GAG GAT GAT GAG GAT GAC TAC TAC GAC GAC 756 GAG GAC GAC GAC GAC GAT GCC TTC TAT GAT GAT GAG GAT GAT 798 GAG GAA GAA GAA TTG GAG AAC CTG ATG GAT GAT GAA TCA GAA 840 GAT GAG GCC GAA GAA GAG ATG AGC GTG GAA ATG GGT GCC GGA 882 GCT GAG GAA ATG GGT GCT GGC GCT AAC TGT GCC TGT GTT CCT 924 GGC CAT CAT TTA AGG AAG AAT GAA GTG AAG TGT AGG ATG ATT 966 TAT TTC TTC CAC GAC CCT AAT TTC CTG GTG TCT ATA CCA GTG 1008 AAC CCT AAG GAA CAA ATG GAG TGT AGG TGT GAA AAT GCT GAT 1050 GAA GAG GTT GCA ATG GAA GAG GAA GAA GAA GAA GAG GAG GAG 1092 GAG GAG GAA GAG GAA ATG GGA AAC CCG GAT GGC TTC TCA CCT 1134 TAG 1137 GCATGCAGTT GCAAAGCCCA GAAGAAAGAA ATGGACAGCG GAAGAAGTGG 1187 TTGTTTTTTT TTCCCCTTCA TTAATTTTCT AGTTTTTAGT AATCCAGAAA 1237 ATTTGATTTT GTTCTAAAGT TCATTATGCA AAGATGTCAC CAACAGACTT 1287 CTGACTGCAT GGTGAACTTT CATATGATAC ATAGGATTAC ACTTGTACCT 1337 GTTAAAAATA AAAGTTTGAC TTGCATAC 1365 4698 base pairs nucleic acid single linear genomic DNA 5 ACCACAGGAG AATGAAAAGA ACCCGGGACT CCCAAAGACG CTAGATGTGT 50 GAAGATCCTG ATCACTCATT GGGTGTCTGA GTTCTGCGAT ATTCATCCCT 100 CAGCCAATGA GCTTACTGTT CTCGTGGGGG GTTTGTGAGC CTTGGGTAGG 150 AAGTTTTGCA AGTTCCGCCT ACAGCTCTAG CTTGTGAATT TGTACCCTTT 200 CACGTAAAAA AGTAGTCCAG AGTTTACTAC ACCCTCCCTC CCCCCTCCCA 250 CCTCGTGCTG TGCTGAGTTT AGAAGTCTTC CTTATAGAAG TCTTCCGTAT 300 AGAACTCTTC CGGAGGAAGG AGGGAGGACC CCCCCCCTTT GCTCTCCCAG 350 CATGCATTGT GTCAACGCCA TTGCACTGAG CTGGTCGAAG AAGTAAGCCG 400 CTAGCTTGCG ACTCTACTCT TATCTTAACT TAGCTCGGCT TCCTGCTGGT 450 ACCCTTTGTG CC 462 ATG TCT GAT AAC AAG AAA CCA GAC AAA GCC CAC AGT GGC TCA 504 GGT GGT GAC GGT GAT GGG AAT AGG TGC AAT TTA TTG CAC CGG 546 TAC TCC CTG GAA GAA ATT CTG CCT TAT CTA GGG TGG CTG GTC 588 TTC GCT GTT GTC ACA ACA AGT TTT CTG GCG CTC CAG ATG TTC 630 ATA GAC GCC CTT TAT GAG GAG CAG TAT GAA AGG GAT GTG GCC 672 TGG ATA GCC AGG CAA AGC AAG CGC ATG TCC TCT GTC GAT GAG 714 GAT GAA GAC GAT GAG GAT GAT GAG GAT GAC TAC TAC GAC GAC 756 GAG GAC GAC GAC GAC GAT GCC TTC TAT GAT GAT GAG GAT GAT 798 GAG GAA GAA GAA TTG GAG AAC CTG ATG GAT GAT GAA TCA GAA 840 GAT GAG GCC GAA GAA GAG ATG AGC GTG GAA ATG GGT GCC GGA 882 GCT GAG GAA ATG GGT GCT GGC GCT AAC TGT GCC T 916 GTGAGTAACC CGTGGTCTTT ACTCTAGATT CAGGTGGGGT GCATTCTTTA 966 CTCTTGCCCA CATCTGTAGT AAAGACCACA TTTTGGTTGG GGGTCATTGC 1016 TGGAGCCATT CCTGGCTCTC CTGTCCACGC CTATCCCCGC TCCTCCCATC 1066 CCCCACTCCT TGCTCCGCTC TCTTTCCTTT TCCCACCTTG CCTCTGGAGC 1116 TTCAGTCCAT CCTGCTCTGC TCCCTTTCCC CTTTGCTCTC CTTGCTCCCC 1166 TCCCCCTCGG CTCAACTTTT CGTGCCTTCT GCTCTCTGAT CCCCACCCTC 1216 TTCAGGCTTC CCCATTTGCT CCTCTCCCGA AACCCTCCCC TTCCTGTTCC 1266 CCTTTTCGCG CCTTTTCTTT CCTGCTCCCC TCCCCCTCCC TATTTACCTT 1316 TCACCAGCTT TGCTCTCCCT GCTCCCCTCC CCCTTTTGCA CCTTTTCTTT 1366 TCCTGCTCCC CTCCCCCTCC CCTCCCTGTT TACCCTTCAC CGCTTTTCCT 1416 CTACCTGCTT CCCTCCCCCT TGCTGCTCCC TCCCTATTTG CATTTTCGGG 1466 TGCTCCTCCC TCCCCCTCCC CCTCCCTCCC TATTTGCATT TTCGGGTGCT 1516 CCTCCCTCCC CCTCCCCAGG CCTTTTTTTT TTTTTTTTTT TTTTTTTTTT 1566 TTGGTTTTTC GAGACAGGGT TTCTCTTTGT ATCCCTGGCT GTCCTGGCAC 1616 TCACTCTGTA GACCAGGCTG GCCTCAAACT CAGAAATCTG CCTGCCTCTG 1666 CCTCCCAAAT GCTGGGATTA AAGGCTTGCA CCAGGACTGC CCCAGTGCAG 1716 GCCTTTCTTT TTTCTCCTCT CTGGTCTCCC TAATCCCTTT TCTGCATGTT 1766 AACTCCCCTT TTGGCACCTT TCCTTTACAG GACCCCCTCC CCCTCCCTGT 1816 TTCCCTTCCG GCACCCTTCC TAGCCCTGCT CTGTTCCCTC TCCCTGCTCC 1866 CCTCCCCCTC TTTGCTCGAC TTTTAGCAGC CTTACCTCTC CCTGCTTTCT 1916 GCCCCGTTCC CCTTTTTTGT GCCTTTCCTC CTGGCTCCCC TCCACCTTCC 1966 AGCTCACCTT TTTGTTTGTT TGGTTGTTTG GTTGTTTGGT TTGCTTTTTT 2016 TTTTTTTTTT GCACCTTGTT TTCCAAGATC CCCCTCCCCC TCCGGCTTCC 2066 CCTCTGTGTG CCTTTCCTGT TCCCTCCCCC TCGCTGGCTC CCCCTCCCTT 2116 TCTGCCTTTC CTGTCCCTGC TCCCTTCTCT GCTAACCTTT TAATGCCTTT 2166 CTTTTCTAGA CTCCCCCCTC CAGGCTTGCT GTTTGCTTCT GTGCACTTTT 2216 CCTGACCCTG CTCCCCTTCC CCTCCCAGCT CCCCCCTCTT TTCCCACCTC 2266 CCTTTCTCCA GCCTGTCACC CCTCCTTCTC TCCTCTCTGT TTCTCCCACT 2316 TCCTGCTTCC TTTACCCCTT CCCTCTCCCT ACTCTCCTCC CTGCCTGCTG 2366 GACTTCCTCT CCAGCCGCCC AGTTCCCTGC AGTCCTGGAG TCTTTCCTGC 2416 CTCTCTGTCC ATCACTTCCC CCTAGTTTCA CTTCCCTTTC ACTCTCCCCT 2466 ATGTGTCTCT CTTCCTATCT ATCCCTTCCT TTCTGTCCCC TCTCCTCTGT 2516 CCATCACCTC TCTCCTCCCT TCCCTTTCCT CTCTCTTCCA TTTTCTTCCA 2566 CCTGCTTCTT TACCCTGCCT CTCCCATTGC CCTCTTACCT TTATGCCCAT 2616 TCCATGTCCC CTCTCAATTC CCTGTCCCAT TGTGCTCCCT CACATCTTCC 2666 ATTTCCCTCT TTCTCCCTTA GCCTCTTCTT CCTCTTCTCT TGTATCTCCC 2716 TTCCCTTTGC TTCTCCCTCC TCCTTTCCCC TTCCCCTATG CCCTCTACTC 2766 TACTTGATCT TCTCTCCTCT CCACATACCC TTTTTCCTTT CCACCCTGCC 2816 CTTTGTCCCC AGACCCTACA GTATCCTGTG CACAGGAAGT GGGAGGTGCC 2866 ATCAACAACA AGGAGGCAAG AAACAGAGCA AAATCCCAAA ATCAGCAGGA 2916 AAGGCTGGAT GAAAATAAGG CCAGGTTCTG AGGACAGCTG GAATCTAGCC 2966 AAGTGGCTCC TATAACCCTA AGTACCAAGG GAGAAAGTGA TGGTGAAGTT 3016 CTTGATCCTT GCTGCTTCTT TTACATATGT TGGCACATCT TTCTCAAATG 3066 CAGGCCATGC TCCATGCTTG GCGCTTGCTC AGCGTGGTTA AGTAATGGGA 3116 GAATCTGAAA ACTAGGGGCC AGTGGTTTGT TTTGGGGACA AATTAGCACG 3166 TAGTGATATT TCCCCCTAAA AATTATAACA AACAGATTCA TGATTTGAGA 3216 TCCTTCTACA GGTGAGAAGT GGAAAAATTG TCACTATGAA GTTCTTTTTA 3266 GGCTAAAGAT ACTTGGAACC ATAGAAGCGT TGTTAAAATA CTGCTTTCTT 3316 TTGCTAAAAT ATTCTTTCTC ACATATTCAT ATTCTCCAG 3355 GT GTT CCT GGC CAT CAT TTA AGG AAG AAT GAA GTG AAG TGT 3396 AGG ATG ATT TAT TTC TTC CAC GAC CCT AAT TTC CTG GTG TCT 3438 ATA CCA GTG AAC CCT AAG GAA CAA ATG GAG TGT AGG TGT GAA 3480 AAT GCT GAT GAA GAG GTT GCA ATG GAA GAG GAA GAA GAA GAA 3522 GAG GAG GAG GAG GAG GAA GAG GAA ATG GGA AAC CCG GAT GGC 3564 TTC TCA CCT TAG 3576 GCATGCAGGT ACTGGCTTCA CTAACCAACC ATTCCTAACA TATGCCTGTA 3626 GCTAAGAGCA TCTTTTTAAA AAATATTATT GGTAAACTAA ACAATTGTTA 3676 TCTTTTTACA TTAATAAGTA TTAAATTAAT CCAGTATACA GTTTTAAGAA 3726 CCCTAAGTTA AACAGAAGTC AATGATGTCT AGATGCCTGT TCTTTAGATT 3776 GTAGTGAGAC TACTTACTAC AGATGAGAAG TTGTTAGACT CGGGAGTAGA 3826 GACCAGTAAA AGATCATGCA GTGAAATGTG GCCATGGAAA TCGCATATTG 3876 TTCTTATAGT ACCTTTGAGA CAGCTGATAA CAGCTGACAA AAATAAGTGT 3926 TTCAAGAAAG ATCACACGCC ATGGTTCACA TGCAAATTAT TATTTTGTCG 3976 TTCTGATTTT TTTCATTTCT AGACCTGTGG TTTTAAAGAG ATGAAAATCT 4026 CTTAAAATTT CCTTCATCTT TAATTTTCCT TAACTTTAGT TTTTTTCACT 4076 TAGAATTCAA TTCAAATTCT TAATTCAATC TTAATTTTTA GATTTCTTAA 4126 AATGTTTTTT AAAAAAAATG CAAATCTCAT TTTTAAGAGA TGAAAGCAGA 4176 GTAACTGGGG GGCTTAGGGA ATCTGTAGGG TTGCGGTATA GCAATAGGGA 4226 GTTCTGGTCT CTGAGAAGCA GTCAGAGAGA ATGGAAAACC AGGCCCTTGC 4276 CAGTAGGTTA GTGAGGTTGA TATGATCAGA TTATGGACAC TCTCCAAATC 4326 ATAAATACTC TAACAGCTAA GGATCTCTGA GGGAAACACA ACAGGGAAAT 4376 ATTTTAGTTT CTCCTTGAGA AACAATGACA AGACATAAAA TTGGCAAGAA 4426 AGTCAGGAGT GTATTCTAAT AAGTGTTGCT TATCTCTTAT TTTCTTCTAC 4476 AGTTGCAAAG CCCAGAAGAA AGAAATGGAC AGCGGAAGAA GTGGTTGTTT 4526 TTTTTTCCCC TTCATTAATT TTCTAGTTTT TAGTAATCCA GAAAATTTGA 4576 TTTTGTTCTA AAGTTCATTA TGCAAAGATG TCACCAACAG ACTTCTGACT 4626 GCATGGTGAA CTTTCATATG ATACATAGGA TTACACTTGT ACCTGTTAAA 4676 AATAAAAGTT TGACTTGCAT AC 4698 9 amino acids amino acid linear protein 6 Leu Pro Tyr Leu Gly Trp Leu Val Phe 5 2419 base pairs nucleic acid single linear genomic DNA 7 GGATCCAGGC CCTGCCAGGA AAAATATAAG GGCCCTGCGT GAGAACAGAG 50 GGGGTCATCC ACTGCATGAG AGTGGGGATG TCACAGAGTC CAGCCCACCC 100 TCCTGGTAGC ACTGAGAAGC CAGGGCTGTG CTTGCGGTCT GCACCCTGAG 150 GGCCCGTGGA TTCCTCTTCC TGGAGCTCCA GGAACCAGGC AGTGAGGCCT 200 TGGTCTGAGA CAGTATCCTC AGGTCACAGA GCAGAGGATG CACAGGGTGT 250 GCCAGCAGTG AATGTTTGCC CTGAATGCAC ACCAAGGGCC CCACCTGCCA 300 CAGGACACAT AGGACTCCAC AGAGTCTGGC CTCACCTCCC TACTGTCAGT 350 CCTGTAGAAT CGACCTCTGC TGGCCGGCTG TACCCTGAGT ACCCTCTCAC 400 TTCCTCCTTC AGGTTTTCAG GGGACAGGCC AACCCAGAGG ACAGGATTCC 450 CTGGAGGCCA CAGAGGAGCA CCAAGGAGAA GATCTGTAAG TAGGCCTTTG 500 TTAGAGTCTC CAAGGTTCAG TTCTCAGCTG AGGCCTCTCA CACACTCCCT 550 CTCTCCCCAG GCCTGTGGGT CTTCATTGCC CAGCTCCTGC CCACACTCCT 600 GCCTGCTGCC CTGACGAGAG TCATCATGTC TCTTGAGCAG AGGAGTCTGC 650 ACTGCAAGCC TGAGGAAGCC CTTGAGGCCC AACAAGAGGC CCTGGGCCTG 700 GTGTGTGTGC AGGCTGCCAC CTCCTCCTCC TCTCCTCTGG TCCTGGGCAC 750 CCTGGAGGAG GTGCCCACTG CTGGGTCAAC AGATCCTCCC CAGAGTCCTC 800 AGGGAGCCTC CGCCTTTCCC ACTACCATCA ACTTCACTCG ACAGAGGCAA 850 CCCAGTGAGG GTTCCAGCAG CCGTGAAGAG GAGGGGCCAA GCACCTCTTG 900 TATCCTGGAG TCCTTGTTCC GAGCAGTAAT CACTAAGAAG GTGGCTGATT 950 TGGTTGGTTT TCTGCTCCTC AAATATCGAG CCAGGGAGCC AGTCACAAAG 1000 GCAGAAATGC TGGAGAGTGT CATCAAAAAT TACAAGCACT GTTTTCCTGA 1050 GATCTTCGGC AAAGCCTCTG AGTCCTTGCA GCTGGTCTTT GGCATTGACG 1100 TGAAGGAAGC AGACCCCACC GGCCACTCCT ATGTCCTTGT CACCTGCCTA 1150 GGTCTCTCCT ATGATGGCCT GCTGGGTGAT AATCAGATCA TGCCCAAGAC 1200 AGGCTTCCTG ATAATTGTCC TGGTCATGAT TGCAATGGAG GGCGGCCATG 1250 CTCCTGAGGA GGAAATCTGG GAGGAGCTGA GTGTGATGGA GGTGTATGAT 1300 GGGAGGGAGC ACAGTGCCTA TGGGGAGCCC AGGAAGCTGC TCACCCAAGA 1350 TTTGGTGCAG GAAAAGTACC TGGAGTACGG CAGGTGCCGG ACAGTGATCC 1400 CGCACGCTAT GAGTTCCTGT GGGGTCCAAG GGCCCTCGCT GAAACCAGCT 1450 ATGTGAAAGT CCTTGAGTAT GTGATCAAGG TCAGTGCAAG AGTTCGCTTT 1500 TTCTTCCCAT CCCTGCGTGA AGCAGCTTTG AGAGAGGAGG AAGAGGGAGT 1550 CTGAGCATGA GTTGCAGCCA AGGCCAGTGG GAGGGGGACT GGGCCAGTGC 1600 ACCTTCCAGG GCCGCGTCCA GCAGCTTCCC CTGCCTCGTG TGACATGAGG 1650 CCCATTCTTC ACTCTGAAGA GAGCGGTCAG TGTTCTCAGT AGTAGGTTTC 1700 TGTTCTATTG GGTGACTTGG AGATTTATCT TTGTTCTCTT TTGGAATTGT 1750 TCAAATGTTT TTTTTTAAGG GATGGTTGAA TGAACTTCAG CATCCAAGTT 1800 TATGAATGAC AGCAGTCACA CAGTTCTGTG TATATAGTTT AAGGGTAAGA 1850 GTCTTGTGTT TTATTCAGAT TGGGAAATCC ATTCTATTTT GTGAATTGGG 1900 ATAATAACAG CAGTGGAATA AGTACTTAGA AATGTGAAAA ATGAGCAGTA 1950 AAATAGATGA GATAAAGAAC TAAAGAAATT AAGAGATAGT CAATTCTTGC 2000 CTTATACCTC AGTCTATTCT GTAAAATTTT TAAAGATATA TGCATACCTG 2050 GATTTCCTTG GCTTCTTTGA GAATGTAAGA GAAATTAAAT CTGAATAAAG 2100 AATTCTTCCT GTTCACTGGC TCTTTTCTTC TCCATGCACT GAGCATCTGC 2150 TTTTTGGAAG GCCCTGGGTT AGTAGTGGAG ATGCTAAGGT AAGCCAGACT 2200 CATACCCACC CATAGGGTCG TAGAGTCTAG GAGCTGCAGT CACGTAATCG 2250 AGGTGGCAAG ATGTCCTCTA AAGATGTAGG GAAAAGTGAG AGAGGGGTGA 2300 GGGTGTGGGG CTCCGGGTGA GAGTGGTGGA GTGTCAATGC CCTGAGCTGG 2350 GGCATTTTGG GCTTTGGGAA ACTGCAGTTC CTTCTGGGGG AGCTGATTGT 2400 AATGATCTTG GGTGGATCC 2419 5674 base pairs nucleic acid single linear genomic DNA MAGE-1 gene 8 CCCGGGGCAC CACTGGCATC CCTCCCCCTA CCACCCCCAA TCCCTCCCTT 50 TACGCCACCC ATCCAAACAT CTTCACGCTC ACCCCCAGCC CAAGCCAGGC 100 AGAATCCGGT TCCACCCCTG CTCTCAACCC AGGGAAGCCC AGGTGCCCAG 150 ATGTGACGCC ACTGACTTGA GCATTAGTGG TTAGAGAGAA GCGAGGTTTT 200 CGGTCTGAGG GGCGGCTTGA GATCGGTGGA GGGAAGCGGG CCCAGCTCTG 250 TAAGGAGGCA AGGTGACATG CTGAGGGAGG ACTGAGGACC CACTTACCCC 300 AGATAGAGGA CCCCAAATAA TCCCTTCATG CCAGTCCTGG ACCATCTGGT 350 GGTGGACTTC TCAGGCTGGG CCACCCCCAG CCCCCTTGCT GCTTAAACCA 400 CTGGGGACTC GAAGTCAGAG CTCCGTGTGA TCAGGGAAGG GCTGCTTAGG 450 AGAGGGCAGC GTCCAGGCTC TGCCAGACAT CATGCTCAGG ATTCTCAAGG 500 AGGGCTGAGG GTCCCTAAGA CCCCACTCCC GTGACCCAAC CCCCACTCCA 550 ATGCTCACTC CCGTGACCCA ACCCCCTCTT CATTGTCATT CCAACCCCCA 600 CCCCACATCC CCCACCCCAT CCCTCAACCC TGATGCCCAT CCGCCCAGCC 650 ATTCCACCCT CACCCCCACC CCCACCCCCA CGCCCACTCC CACCCCCACC 700 CAGGCAGGAT CCGGTTCCCG CCAGGAAACA TCCGGGTGCC CGGATGTGAC 750 GCCACTGACT TGCGCATTGT GGGGCAGAGA GAAGCGAGGT TTCCATTCTG 800 AGGGACGGCG TAGAGTTCGG CCGAAGGAAC CTGACCCAGG CTCTGTGAGG 850 AGGCAAGGTG AGAGGCTGAG GGAGGACTGA GGACCCCGCC ACTCCAAATA 900 GAGAGCCCCA AATATTCCAG CCCCGCCCTT GCTGCCAGCC CTGGCCCACC 950 CGCGGGAAGA CGTCTCAGCC TGGGCTGCCC CCAGACCCCT GCTCCAAAAG 1000 CCTTGAGAGA CACCAGGTTC TTCTCCCCAA GCTCTGGAAT CAGAGGTTGC 1050 TGTGACCAGG GCAGGACTGG TTAGGAGAGG GCAGGGCACA GGCTCTGCCA 1100 GGCATCAAGA TCAGCACCCA AGAGGGAGGG CTGTGGGCCC CCAAGACTGC 1150 ACTCCAATCC CCACTCCCAC CCCATTCGCA TTCCCATTCC CCACCCAACC 1200 CCCATCTCCT CAGCTACACC TCCACCCCCA TCCCTACTCC TACTCCGTCA 1250 CCTGACCACC ACCCTCCAGC CCCAGCACCA GCCCCAACCC TTCTGCCACC 1300 TCACCCTCAC TGCCCCCAAC CCCACCCTCA TCTCTCTCAT GTGCCCCACT 1350 CCCATCGCCT CCCCCATTCT GGCAGAATCC GGTTTGCCCC TGCTCTCAAC 1400 CCAGGGAAGC CCTGGTAGGC CCGATGTGAA ACCACTGACT TGAACCTCAC 1450 AGATCTGAGA GAAGCCAGGT TCATTTAATG GTTCTGAGGG GCGGCTTGAG 1500 ATCCACTGAG GGGAGTGGTT TTAGGCTCTG TGAGGAGGCA AGGTGAGATG 1550 CTGAGGGAGG ACTGAGGAGG CACACACCCC AGGTAGATGG CCCCAAAATG 1600 ATCCAGTACC ACCCCTGCTG CCAGCCCTGG ACCACCCGGC CAGGACAGAT 1650 GTCTCAGCTG GACCACCCCC CGTCCCGTCC CACTGCCACT TAACCCACAG 1700 GGCAATCTGT AGTCATAGCT TATGTGACCG GGGCAGGGTT GGTCAGGAGA 1750 GGCAGGGCCC AGGCATCAAG GTCCAGCATC CGCCCGGCAT TAGGGTCAGG 1800 ACCCTGGGAG GGAACTGAGG GTTCCCCACC CACACCTGTC TCCTCATCTC 1850 CACCGCCACC CCACTCACAT TCCCATACCT ACCCCCTACC CCCAACCTCA 1900 TCTTGTCAGA ATCCCTGCTG TCAACCCACG GAAGCCACGG GAATGGCGGC 1950 CAGGCACTCG GATCTTGACG TCCCCATCCA GGGTCTGATG GAGGGAAGGG 2000 GCTTGAACAG GGCCTCAGGG GAGCAGAGGG AGGGCCCTAC TGCGAGATGA 2050 GGGAGGCCTC AGAGGACCCA GCACCCTAGG ACACCGCACC CCTGTCTGAG 2100 ACTGAGGCTG CCACTTCTGG CCTCAAGAAT CAGAACGATG GGGACTCAGA 2150 TTGCATGGGG GTGGGACCCA GGCCTGCAAG GCTTACGCGG AGGAAGAGGA 2200 GGGAGGACTC AGGGGACCTT GGAATCCAGA TCAGTGTGGA CCTCGGCCCT 2250 GAGAGGTCCA GGGCACGGTG GCCACATATG GCCCATATTT CCTGCATCTT 2300 TGAGGTGACA GGACAGAGCT GTGGTCTGAG AAGTGGGGCC TCAGGTCAAC 2350 AGAGGGAGGA GTTCCAGGAT CCATATGGCC CAAGATGTGC CCCCTTCATG 2400 AGGACTGGGG ATATCCCCGG CTCAGAAAGA AGGGACTCCA CACAGTCTGG 2450 CTGTCCCCTT TTAGTAGCTC TAGGGGGACC AGATCAGGGA TGGCGGTATG 2500 TTCCATTCTC ACTTGTACCA CAGGCAGGAA GTTGGGGGGC CCTCAGGGAG 2550 ATGGGGTCTT GGGGTAAAGG GGGGATGTCT ACTCATGTCA GGGAATTGGG 2600 GGTTGAGGAA GCACAGGCGC TGGCAGGAAT AAAGATGAGT GAGACAGACA 2650 AGGCTATTGG AATCCACACC CCAGAACCAA AGGGGTCAGC CCTGGACACC 2700 TCACCCAGGA TGTGGCTTCT TTTTCACTCC TGTTTCCAGA TCTGGGGCAG 2750 GTGAGGACCT CATTCTCAGA GGGTGACTCA GGTCAACGTA GGGACCCCCA 2800 TCTGGTCTAA AGACAGAGCG GTCCCAGGAT CTGCCATGCG TTCGGGTGAG 2850 GAACATGAGG GAGGACTGAG GGTACCCCAG GACCAGAACA CTGAGGGAGA 2900 CTGCACAGAA ATCAGCCCTG CCCCTGCTGT CACCCCAGAG AGCATGGGCT 2950 GGGCCGTCTG CCGAGGTCCT TCCGTTATCC TGGGATCATT GATGTCAGGG 3000 ACGGGGAGGC CTTGGTCTGA GAAGGCTGCG CTCAGGTCAG TAGAGGGAGC 3050 GTCCCAGGCC CTGCCAGGAG TCAAGGTGAG GACCAAGCGG GCACCTCACC 3100 CAGGACACAT TAATTCCAAT GAATTTTGAT ATCTCTTGCT GCCCTTCCCC 3150 AAGGACCTAG GCACGTGTGG CCAGATGTTT GTCCCCTCCT GTCCTTCCAT 3200 TCCTTATCAT GGATGTGAAC TCTTGATTTG GATTTCTCAG ACCAGCAAAA 3250 GGGCAGGATC CAGGCCCTGC CAGGAAAAAT ATAAGGGCCC TGCGTGAGAA 3300 CAGAGGGGGT CATCCACTGC ATGAGAGTGG GGATGTCACA GAGTCCAGCC 3350 CACCCTCCTG GTAGCACTGA GAAGCCAGGG CTGTGCTTGC GGTCTGCACC 3400 CTGAGGGCCC GTGGATTCCT CTTCCTGGAG CTCCAGGAAC CAGGCAGTGA 3450 GGCCTTGGTC TGAGACAGTA TCCTCAGGTC ACAGAGCAGA GGATGCACAG 3500 GGTGTGCCAG CAGTGAATGT TTGCCCTGAA TGCACACCAA GGGCCCCACC 3550 TGCCACAGGA CACATAGGAC TCCACAGAGT CTGGCCTCAC CTCCCTACTG 3600 TCAGTCCTGT AGAATCGACC TCTGCTGGCC GGCTGTACCC TGAGTACCCT 3650 CTCACTTCCT CCTTCAGGTT TTCAGGGGAC AGGCCAACCC AGAGGACAGG 3700 ATTCCCTGGA GGCCACAGAG GAGCACCAAG GAGAAGATCT GTAAGTAGGC 3750 CTTTGTTAGA GTCTCCAAGG TTCAGTTCTC AGCTGAGGCC TCTCACACAC 3800 TCCCTCTCTC CCCAGGCCTG TGGGTCTTCA TTGCCCAGCT CCTGCCCACA 3850 CTCCTGCCTG CTGCCCTGAC GAGAGTCATC 3880 ATG TCT CTT GAG CAG AGG AGT CTG CAC TGC AAG CCT GAG GAA 3922 GCC CTT GAG GCC CAA CAA GAG GCC CTG GGC CTG GTG TGT GTG 3964 CAG GCT GCC ACC TCC TCC TCC TCT CCT CTG GTC CTG GGC ACC 4006 CTG GAG GAG GTG CCC ACT GCT GGG TCA ACA GAT CCT CCC CAG 4048 AGT CCT CAG GGA GCC TCC GCC TTT CCC ACT ACC ATC AAC TTC 4090 ACT CGA CAG AGG CAA CCC AGT GAG GGT TCC AGC AGC CGT GAA 4132 GAG GAG GGG CCA AGC ACC TCT TGT ATC CTG GAG TCC TTG TTC 4174 CGA GCA GTA ATC ACT AAG AAG GTG GCT GAT TTG GTT GGT TTT 4216 CTG CTC CTC AAA TAT CGA GCC AGG GAG CCA GTC ACA AAG GCA 4258 GAA ATG CTG GAG AGT GTC ATC AAA AAT TAC AAG CAC TGT TTT 4300 CCT GAG ATC TTC GGC AAA GCC TCT GAG TCC TTG CAG CTG GTC 4342 TTT GGC ATT GAC GTG AAG GAA GCA GAC CCC ACC GGC CAC TCC 4384 TAT GTC CTT GTC ACC TGC CTA GGT CTC TCC TAT GAT GGC CTG 4426 CTG GGT GAT AAT CAG ATC ATG CCC AAG ACA GGC TTC CTG ATA 4468 ATT GTC CTG GTC ATG ATT GCA ATG GAG GGC GGC CAT GCT CCT 4510 GAG GAG GAA ATC TGG GAG GAG CTG AGT GTG ATG GAG GTG TAT 4552 GAT GGG AGG GAG CAC AGT GCC TAT GGG GAG CCC AGG AAG CTG 4594 CTC ACC CAA GAT TTG GTG CAG GAA AAG TAC CTG GAG TAC GGC 4636 AGG TGC CGG ACA GTG ATC CCG CAC GCT ATG AGT TCC TGT GGG 4678 GTC CAA GGG CCC TCG CTG AAA CCA GCT ATG TGA 4711 AAGTCCTTGA GTATGTGATC AAGGTCAGTG CAAGAGTTC 4750 GCTTTTTCTT CCCATCCCTG CGTGAAGCAG CTTTGAGAGA GGAGGAAGAG 4800 GGAGTCTGAG CATGAGTTGC AGCCAAGGCC AGTGGGAGGG GGACTGGGCC 4850 AGTGCACCTT CCAGGGCCGC GTCCAGCAGC TTCCCCTGCC TCGTGTGACA 4900 TGAGGCCCAT TCTTCACTCT GAAGAGAGCG GTCAGTGTTC TCAGTAGTAG 4950 GTTTCTGTTC TATTGGGTGA CTTGGAGATT TATCTTTGTT CTCTTTTGGA 5000 ATTGTTCAAA TGTTTTTTTT TAAGGGATGG TTGAATGAAC TTCAGCATCC 5050 AAGTTTATGA ATGACAGCAG TCACACAGTT CTGTGTATAT AGTTTAAGGG 5100 TAAGAGTCTT GTGTTTTATT CAGATTGGGA AATCCATTCT ATTTTGTGAA 5150 TTGGGATAAT AACAGCAGTG GAATAAGTAC TTAGAAATGT GAAAAATGAG 5200 CAGTAAAATA GATGAGATAA AGAACTAAAG AAATTAAGAG ATAGTCAATT 5250 CTTGCCTTAT ACCTCAGTCT ATTCTGTAAA ATTTTTAAAG ATATATGCAT 5300 ACCTGGATTT CCTTGGCTTC TTTGAGAATG TAAGAGAAAT TAAATCTGAA 5350 TAAAGAATTC TTCCTGTTCA CTGGCTCTTT TCTTCTCCAT GCACTGAGCA 5400 TCTGCTTTTT GGAAGGCCCT GGGTTAGTAG TGGAGATGCT AAGGTAAGCC 5450 AGACTCATAC CCACCCATAG GGTCGTAGAG TCTAGGAGCT GCAGTCACGT 5500 AATCGAGGTG GCAAGATGTC CTCTAAAGAT GTAGGGAAAA GTGAGAGAGG 5550 GGTGAGGGTG TGGGGCTCCG GGTGAGAGTG GTGGAGTGTC AATGCCCTGA 5600 GCTGGGGCAT TTTGGGCTTT GGGAAACTGC AGTTCCTTCT GGGGGAGCTG 5650 ATTGTAATGA TCTTGGGTGG ATCC 5674 4157 base pairs nucleic acid single linear genomic DNA MAGE-2 gene 9 CCCATCCAGA TCCCCATCCG GGCAGAATCC GGTTCCACCC TTGCCGTGAA 50 CCCAGGGAAG TCACGGGCCC GGATGTGACG CCACTGACTT GCACATTGGA 100 GGTCAGAGGA CAGCGAGATT CTCGCCCTGA GCAACGGCCT GACGTCGGCG 150 GAGGGAAGCA GGCGCAGGCT CCGTGAGGAG GCAAGGTAAG ACGCCGAGGG 200 AGGACTGAGG CGGGCCTCAC CCCAGACAGA GGGCCCCCAA TTAATCCAGC 250 GCTGCCTCTG CTGCCGGGCC TGGACCACCC TGCAGGGGAA GACTTCTCAG 300 GCTCAGTCGC CACCACCTCA CCCCGCCACC CCCCGCCGCT TTAACCGCAG 350 GGAACTCTGG CGTAAGAGCT TTGTGTGACC AGGGCAGGGC TGGTTAGAAG 400 TGCTCAGGGC CCAGACTCAG CCAGGAATCA AGGTCAGGAC CCCAAGAGGG 450 GACTGAGGGC AACCCACCCC CTACCCTCAC TACCAATCCC ATCCCCCAAC 500 ACCAACCCCA CCCCCATCCC TCAAACACCA ACCCCACCCC CAAACCCCAT 550 TCCCATCTCC TCCCCCACCA CCATCCTGGC AGAATCCGGC TTTGCCCCTG 600 CAATCAACCC ACGGAAGCTC CGGGAATGGC GGCCAAGCAC GCGGATCCTG 650 ACGTTCACAT GTACGGCTAA GGGAGGGAAG GGGTTGGGTC TCGTGAGTAT 700 GGCCTTTGGG ATGCAGAGGA AGGGCCCAGG CCTCCTGGAA GACAGTGGAG 750 TCCTTAGGGG ACCCAGCATG CCAGGACAGG GGGCCCACTG TACCCCTGTC 800 TCAAACTGAG CCACCTTTTC ATTCAGCCGA GGGAATCCTA GGGATGCAGA 850 CCCACTTCAG GGGGTTGGGG CCCAGCCTGC GAGGAGTCAA GGGGAGGAAG 900 AAGAGGGAGG ACTGAGGGGA CCTTGGAGTC CAGATCAGTG GCAACCTTGG 950 GCTGGGGGAT CCTGGGCACA GTGGCCGAAT GTGCCCCGTG CTCATTGCAC 1000 CTTCAGGGTG ACAGAGAGTT GAGGGCTGTG GTCTGAGGGC TGGGACTTCA 1050 GGTCAGCAGA GGGAGGAATC CCAGGATCTG CCGGACCCAA GGTGTGCCCC 1100 CTTCATGAGG ACTCCCCATA CCCCCGGCCC AGAAAGAAGG GATGCCACAG 1150 AGTCTGGAAG TAAATTGTTC TTAGCTCTGG GGGAACCTGA TCAGGGATGG 1200 CCCTAAGTGA CAATCTCATT TGTACCACAG GCAGGAGGTT GGGGAACCCT 1250 CAGGGAGATA AGGTGTTGGT GTAAAGAGGA GCTGTCTGCT CATTTCAGGG 1300 GGTTCCCCCT TGAGAAAGGG CAGTCCCTGG CAGGAGTAAA GATGAGTAAC 1350 CCACAGGAGG CCATCATAAC GTTCACCCTA GAACCAAAGG GGTCAGCCCT 1400 GGACAACGCA CGTGGGGTAA CAGGATGTGG CCCCTCCTCA CTTGTCTTTC 1450 CAGATCTCAG GGAGTTGATG ACCTTGTTTT CAGAAGGTGA CTCAGTCAAC 1500 ACAGGGGCCC CTCTGGTCGA CAGATGCAGT GGTTCTAGGA TCTGCCAAGC 1550 ATCCAGGTGG AGAGCCTGAG GTAGGATTGA GGGTACCCCT GGGCCAGAAT 1600 GCAGCAAGGG GGCCCCATAG AAATCTGCCC TGCCCCTGCG GTTACTTCAG 1650 AGACCCTGGG CAGGGCTGTC AGCTGAAGTC CCTCCATTAT CTGGGATCTT 1700 TGATGTCAGG GAAGGGGAGG CCTTGGTCTG AAGGGGCTGG AGTCAGGTCA 1750 GTAGAGGGAG GGTCTCAGGC CCTGCCAGGA GTGGACGTGA GGACCAAGCG 1800 GACTCGTCAC CCAGGACACC TGGACTCCAA TGAATTTGAC ATCTCTCGTT 1850 GTCCTTCGCG GAGGACCTGG TCACGTATGG CCAGATGTGG GTCCCCTCTA 1900 TCTCCTTCTG TACCATATCA GGGATGTGAG TTCTTGACAT GAGAGATTCT 1950 CAAGCCAGCA AAAGGGTGGG ATTAGGCCCT ACAAGGAGAA AGGTGAGGGC 2000 CCTGAGTGAG CACAGAGGGG ACCCTCCACC CAAGTAGAGT GGGGACCTCA 2050 CGGAGTCTGG CCAACCCTGC TGAGACTTCT GGGAATCCGT GGCTGTGCTT 2100 GCAGTCTGCA CACTGAAGGC CCGTGCATTC CTCTCCCAGG AATCAGGAGC 2150 TCCAGGAACC AGGCAGTGAG GCCTTGGTCT GAGTCAGTGC CTCAGGTCAC 2200 AGAGCAGAGG GGACGCAGAC AGTGCCAACA CTGAAGGTTT GCCTGGAATG 2250 CACACCAAGG GCCCCACCCG CCCAGAACAA ATGGGACTCC AGAGGGCCTG 2300 GCCTCACCCT CCCTATTCTC AGTCCTGCAG CCTGAGCATG TGCTGGCCGG 2350 CTGTACCCTG AGGTGCCCTC CCACTTCCTC CTTCAGGTTC TGAGGGGGAC 2400 AGGCTGACAA GTAGGACCCG AGGCACTGGA GGAGCATTGA AGGAGAAGAT 2450 CTGTAAGTAA GCCTTTGTCA GAGCCTCCAA GGTTCAGTTC AGTTCTCACC 2500 TAAGGCCTCA CACACGCTCC TTCTCTCCCC AGGCCTGTGG GTCTTCATTG 2550 CCCAGCTCCT GCCCGCACTC CTGCCTGCTG CCCTGACCAG AGTCATC 2597 ATG CCT CTT GAG CAG AGG AGT CAG CAC TGC AAG CCT GAA GAA 2639 GGC CTT GAG GCC CGA GGA GAG GCC CTG GGC CTG GTG GGT GCG 2681 CAG GCT CCT GCT ACT GAG GAG CAG CAG ACC GCT TCT TCC TCT 2723 TCT ACT CTA GTG GAA GTT ACC CTG GGG GAG GTG CCT GCT GCC 2765 GAC TCA CCG AGT CCT CCC CAC AGT CCT CAG GGA GCC TCC AGC 2807 TTC TCG ACT ACC ATC AAC TAC ACT CTT TGG AGA CAA TCC GAT 2849 GAG GGC TCC AGC AAC CAA GAA GAG GAG GGG CCA AGA ATG TTT 2891 CCC GAC CTG GAG TCC GAG TTC CAA GCA GCA ATC AGT AGG AAG 2933 ATG GTT GAG TTG GTT CAT TTT CTG CTC CTC AAG TAT CGA GCC 2975 AGG GAG CCG GTC ACA AAG GCA GAA ATG CTG GAG AGT GTC CTC 3017 AGA AAT TGC CAG GAC TTC TTT CCC GTG ATC TTC AGC AAA GCC 3059 TCC GAG TAC TTG CAG CTG GTC TTT GGC ATC GAG GTG GTG GAA 3101 GTG GTC CCC ATC AGC CAC TTG TAC ATC CTT GTC ACC TGC CTG 3143 GGC CTC TCC TAC GAT GGC CTG CTG GGC GAC AAT CAG GTC ATG 3185 CCC AAG ACA GGC CTC CTG ATA ATC GTC CTG GCC ATA ATC GCA 3227 ATA GAG GGC GAC TGT GCC CCT GAG GAG AAA ATC TGG GAG GAG 3269 CTG AGT ATG TTG GAG GTG TTT GAG GGG AGG GAG GAC AGT GTC 3311 TTC GCA CAT CCC AGG AAG CTG CTC ATG CAA GAT CTG GTG CAG 3353 GAA AAC TAC CTG GAG TAC CGG CAG GTG CCC GGC AGT GAT CCT 3395 GCA TGC TAC GAG TTC CTG TGG GGT CCA AGG GCC CTC ATT GAA 3437 ACC AGC TAT GTG AAA GTC CTG CAC CAT ACA CTA AAG ATC GGT 3479 GGA GAA CCT CAC ATT TCC TAC CCA CCC CTG CAT GAA CGG GCT 3521 TTG AGA GAG GGA GAA GAG TGA 3542 GTCTCAGCAC ATGTTGCAGC CAGGGCCAGT GGGAGGGGGT CTGGGCCAGT 3592 GCACCTTCCA GGGCCCCATC CATTAGCTTC CACTGCCTCG TGTGATATGA 3642 GGCCCATTCC TGCCTCTTTG AAGAGAGCAG TCAGCATTCT TAGCAGTGAG 3692 TTTCTGTTCT GTTGGATGAC TTTGAGATTT ATCTTTCTTT CCTGTTGGAA 3742 TTGTTCAAAT GTTCCTTTTA ACAAATGGTT GGATGAACTT CAGCATCCAA 3792 GTTTATGAAT GACAGTAGTC ACACATAGTG CTGTTTATAT AGTTTAGGGG 3842 TAAGAGTCCT GTTTTTTATT CAGATTGGGA AATCCATTCC ATTTTGTGAG 3892 TTGTCACATA ATAACAGCAG TGGAATATGT ATTTGCCTAT ATTGTGAACG 3942 AATTAGCAGT AAAATACATG ATACAAGGAA CTCAAAAGAT AGTTAATTCT 3992 TGCCTTATAC CTCAGTCTAT TATGTAAAAT TAAAAATATG TGTATGTTTT 4042 TGCTTCTTTG AGAATGCAAA AGAAATTAAA TCTGAATAAA TTCTTCCTGT 4092 TCACTGGCTC ATTTCTTTAC CATTCACTCA GCATCTGCTC TGTGGAAGGC 4142 CCTGGTAGTA GTGGG 4157 662 base pairs nucleic acid single linear genomic DNA MAGE-21 gene 10 GGATCCCCAT GGATCCAGGA AGAATCCAGT TCCACCCCTG CTGTGAACCC 50 AGGGAAGTCA CGGGGCCGGA TGTGACGCCA CTGACTTGCG CGTTGGAGGT 100 CAGAGAACAG CGAGATTCTC GCCCTGAGCA ACGGCCTGAC GTCGGCGGAG 150 GGAAGCAGGC GCAGGCTCCG TGAGGAGGCA AGGTAAGATG CCGAGGGAGG 200 ACTGAGGCGG GCCTCACCCC AGACAGAGGG CCCCCAATAA TCCAGCGCTG 250 CCTCTGCTGC CAGGCCTGGA CCACCCTGCA GGGGAAGACT TCTCAGGCTC 300 AGTCGCCACC ACCTCACCCC GCCACCCCCC GCCGCTTTAA CCGCAGGGAA 350 CTCTGGTGTA AGAGCTTTGT GTGACCAGGG CAGGGCTGGT TAGAAGTGCT 400 CAGGGCCCAG ACTCAGCCAG GAATCAAGGT CAGGACCCCA AGAGGGGACT 450 GAGGGTAACC CCCCCGCACC CCCACCACCA TTCCCATCCC CCAACACCAA 500 CCCCACCCCC ATCCCCCAAC ACCAAACCCA CCACCATCGC TCAAACATCA 550 ACGGCACCCC CAAACCCCGA TTCCCATCCC CACCCATCCT GGCAGAATCG 600 GAGCTTTGCC CCTGCAATCA ACCCACGGAA GCTCCGGGAA TGGCGGCCAA 650 GCACGCGGAT CC 662 1640 base pairs nucleic acid single linear cDNA to mRNA cDNA MAGE-3 11 GCCGCGAGGG AAGCCGGCCC AGGCTCGGTG AGGAGGCAAG GTTCTGAGGG 50 GACAGGCTGA CCTGGAGGAC CAGAGGCCCC CGGAGGAGCA CTGAAGGAGA 100 AGATCTGCCA GTGGGTCTCC ATTGCCCAGC TCCTGCCCAC ACTCCCGCCT 150 GTTGCCCTGA CCAGAGTCAT C 171 ATG CCT CTT GAG CAG AGG AGT CAG CAC TGC AAG CCT GAA GAA 213 GGC CTT GAG GCC CGA GGA GAG GCC CTG GGC CTG GTG GGT GCG 255 CAG GCT CCT GCT ACT GAG GAG CAG GAG GCT GCC TCC TCC TCT 297 TCT ACT CTA GTT GAA GTC ACC CTG GGG GAG GTG CCT GCT GCC 339 GAG TCA CCA GAT CCT CCC CAG AGT CCT CAG GGA GCC TCC AGC 381 CTC CCC ACT ACC ATG AAC TAC CCT CTC TGG AGC CAA TCC TAT 423 GAG GAC TCC AGC AAC CAA GAA GAG GAG GGG CCA AGC ACC TTC 465 CCT GAC CTG GAG TCC GAG TTC CAA GCA GCA CTC AGT AGG AAG 507 GTG GCC GAG TTG GTT CAT TTT CTG CTC CTC AAG TAT CGA GCC 549 AGG GAG CCG GTC ACA AAG GCA GAA ATG CTG GGG AGT GTC GTC 591 GGA AAT TGG CAG TAT TTC TTT CCT GTG ATC TTC AGC AAA GCT 633 TCC AGT TCC TTG CAG CTG GTC TTT GGC ATC GAG CTG ATG GAA 675 GTG GAC CCC ATC GGC CAC TTG TAC ATC TTT GCC ACC TGC CTG 717 GGC CTC TCC TAC GAT GGC CTG CTG GGT GAC AAT CAG ATC ATG 759 CCC AAG GCA GGC CTC CTG ATA ATC GTC CTG GCC ATA ATC GCA 801 AGA GAG GGC GAC TGT GCC CCT GAG GAG AAA ATC TGG GAG GAG 843 CTG AGT GTG TTA GAG GTG TTT GAG GGG AGG GAA GAC AGT ATG 885 TTG GGG GAT CCC AAG AAG CTG CTC ACC CAA CAT TTC GTG CAG 927 GAA AAC TAC CTG GAG TAC CGG CAG GTC CCC GGC AGT GAT CCT 969 GCA TGT TAT GAA TTC CTG TGG GGT CCA AGG GCC CTC GTT GAA 1011 ACC AGC TAT GTG AAA GTC CTG CAC CAT ATG GTA AAG ATC AGT 1053 GGA GGA CCT CAC ATT TCC TAC CCA CCC CTG CAT GAG TGG GTT 1095 TTG AGA GAG GGG GAA GAG TGA 1116 GTCTGAGCAC GAGTTGCAGC CAGGGCCAGT GGGAGGGGGT CTGGGCCAGT 1166 GCACCTTCCG GGGCCGCATC CCTTAGTTTC CACTGCCTCC TGTGACGTGA 1216 GGCCCATTCT TCACTCTTTG AAGCGAGCAG TCAGCATTCT TAGTAGTGGG 1266 TTTCTGTTCT GTTGGATGAC TTTGAGATTA TTCTTTGTTT CCTGTTGGAG 1316 TTGTTCAAAT GTTCCTTTTA ACGGATGGTT GAATGAGCGT CAGCATCCAG 1366 GTTTATGAAT GACAGTAGTC ACACATAGTG CTGTTTATAT AGTTTAGGAG 1416 TAAGAGTCTT GttTTTTACT CAAATTgGGA AATCCATTCC ATTTTGTGAA 1466 TTGTGACATA ATAATAGCAG TGGTAAAAGT ATTTGCTTAA AATTGTGAGC 1516 GAATTAGCAA TAACATACAT GAGATAACTC AAGAAATCAA AAGATAGTTG 1566 ATTCTTGCCT TGTACCTCAA TCTATTCTGT AAAATTAAAC AAATATGCAA 1616 ACCAGGATTT CCTTGACTTC TTTG 1640 943 base pairs nucleic acid single linear genomic DNA MAGE-31 gene 12 GGATCCTCCA CCCCAGTAGA GTGGGGACCT CACAGAGTCT GGCCAACCCT 50 CCTGACAGTT CTGGGAATCC GTGGCTGCGT TTGCTGTCTG CACATTGGGG 100 GCCCGTGGAT TCCTCTCCCA GGAATCAGGA GCTCCAGGAA CAAGGCAGTG 150 AGGACTTGGT CTGAGGCAGT GTCCTCAGGT CACAGAGTAG AGGGGgCTCA 200 GATAGTGCCA ACGGTGAAGG TTTGCCTTGG ATTCAAACCA AGGGCCCCAC 250 CTGCCCCAGA ACACATGGAC TCCAGAGCGC CTGGCCTCAC CCTCAATACT 300 TTCAGTCCTG CAGCCTCAGC ATGCGCTGGC CGGATGTACC CTGAGGTGCC 350 CTCTCACTTC CTCCTTCAGG TTCTGAGGGG ACAGGCTGAC CTGGAGGACC 400 AGAGGCCCCC GGAGGAGCAC TGAAGGAGAA GATCTGTAAG TAAGCCTTTG 450 TTAGAGCCTC CAAGGTTCCA TTCAGTACTC AGCTGAGGTC TCTCACATGC 500 TCCCTCTCTC CCCAGGCCAG TGGGTCTCCA TTGCCCAGCT CCTGCCCACA 550 CTCCCGCCTG TTGCCCTGAC CAGAGTCATC 580 ATG CCT CTT GAG CAG AGG AGT CAG CAC TGC AAG CCT GAA GAA 622 GGC CTT GAG GCC CGA GGA GAg GCC CTG GGC CTG GTG GGT GCG 664 CAG GCT CCT GCT ACT GAG GAG CAG GAG GCT GCC TCC TCC TCT 706 TCT AGT GTA GTT GAA GTC ACC CTG GGG GAG GTG CCT GCT GCC 748 GAG TCA CCA GAT CCT CCC CAG AGT CCT CAG GGA GCC TCC AGC 790 CTC CCC ACT ACC ATG AAC TAC CCT CTC TGG AGC CAA TCC TAT 832 GAG GAC TCC AGC AAC CAA GAA GAG GAG GGG CCA AGC ACC TTC 874 CCT GAC CTG GAG TCT GAG TTC CAA GCA GCA CTC AGT AGG AAG 916 GTG GCC AAG TTG GTT CAT TTT CTG CTC 943 2531 base pairs nucleic acid single linear genomic DNA MAGE-4 gene 13 GGATCCAGGC CCTGCCTGGA GAAATGTGAG GGCCCTGAGT GAACACAGTG 50 GGGATCATCC ACTCCATGAG AGTGGGGACC TCACAGAGTC CAGCCTACCC 100 TCTTGATGGC ACTGAGGGAC CGGGGCTGTG CTTACAGTCT GCACCCTAAG 150 GGCCCATGGA TTCCTCTCCT AGGAGCTCCA GGAACAAGGC AGTGAGGCCT 200 TGGTCTGAGA CAGTGTCCTC AGGTTACAGA GCAGAGGATG CACAGGCTGT 250 GCCAGCAGTG AATGTTTGCC CTGAATGCAC ACCAAGGGCC CCACCTGCCA 300 CAAGACACAT AGGACTCCAA AGAGTCTGGC CTCACCTCCC TACCATCAAT 350 CCTGCAGAAT CGACCTCTGC TGGCCGGCTA TACCCTGAGG TGCTCTCTCA 400 CTTCCTCCTT CAGGTTCTGA GCAGACAGGC CAACCGGAGA CAGGATTCCC 450 TGGAGGCCAC AGAGGAGCAC CAAGGAGAAG ATCTGTAAGT AAGCCTTTGT 500 TAGAGCCTCT AAGATTTGGT TCTCAGCTGA GGTCTCTCAC ATGCTCCCTC 550 TCTCCGTAGG CCTGTGGGTC CCCATTGCCC AGCTTTTGCC TGCACTCTTG 600 CCTGCTGCCC TGACCAGAGT CATC 624 ATG TCT TCT GAG CAG AAG AGT CAG CAC TGC AAG CCT GAG GAA 666 GGC GTT GAG GCC CAA GAA GAG GCC CTG GGC CTG GTG GGT GCA 708 CAG GCT CCT ACT ACT GAG GAG CAG GAG GCT GCT GTC TCC TCC 750 TCC TCT CCT CTG GTC CCT GGC ACC CTG GAG GAA GTG CCT GCT 792 GCT GAG TCA GCA GGT CCT CCC CAG AGT CCT CAG GGA GCC TCT 834 GCC TTA CCC ACT ACC ATC AGC TTC ACT TGC TGG AGG CAA CCC 876 AAT GAG GGT TCC AGC AGC CAA GAA GAG GAG GGG CCA AGC ACC 918 TCG CCT GAC GCA GAG TCC TTG TTC CGA GAA GCA CTC AGT AAC 960 AAG GTG GAT GAG TTG GCT CAT TTT CTG CTC CGC AAG TAT CGA 1002 GCC AAG GAG CTG GTC ACA AAG GCA GAA ATG CTG GAG AGA GTC 1044 ATC AAA AAT TAC AAG CGC TGC TTT CCT GTG ATC TTC GGC AAA 1086 GCC TCC GAG TCC CTG AAG ATG ATC TTT GGC ATT GAC GTG AAG 1128 GAA GTG GAC CCC GCC AGC AAC ACC TAC ACC CTT GTC ACC TGC 1170 CTG GGC CTT TCC TAT GAT GGC CTG CTG GGT AAT AAT CAG ATC 1212 TTT CCC AAG ACA GGC CTT CTG ATA ATC GTC CTG GGC ACA ATT 1254 GCA ATG GAG GGC GAC AGC GCC TCT GAG GAG GAA ATC TGG GAG 1296 GAG CTG GGT GTG ATG GGG GTG TAT GAT GGG AGG GAG CAC ACT 1338 GTC TAT GGG GAG CCC AGG AAA CTG CTC ACC CAA GAT TGG GTG 1380 CAG GAA AAC TAC CTG GAG TAC CGG CAG GTA CCC GGC AGT AAT 1422 CCT GCG CGC TAT GAG TTC CTG TGG GGT CCA AGG GCT CTG GCT 1464 GAA ACC AGC TAT GTG AAA GTC CTG GAG CAT GTG GTC AGG GTC 1506 AAT GCA AGA GTT CGC ATT GCC TAC CCA TCC CTG CGT GAA GCA 1548 GCT TTG TTA GAG GAG GAA GAG GGA GTC TGA 1578 GCATGAGTTG CAGCCAGGGC TGTGGGGAAG GGGCAGGGCT GGGCCAGTGC 1628 ATCTAACAGC CCTGTGCAGC AGCTTCCCTT GCCTCGTGTA ACATGAGGCC 1678 CATTCTTCAC TCTGTTTGAA GAAAATAGTC AGTGTTCTTA GTAGTGGGTT 1728 TCTATTTTGT TGGATGACTT GGAGATTTAT CTCTGTTTCC TTTTACAATT 1778 GTTGAAATGT TCCTTTTAAT GGATGGTTGA ATTAACTTCA GCATCCAAGT 1828 TTATGAATCG TAGTTAACGT ATATTGCTGT TAATATAGTT TAGGAGTAAG 1878 AGTCTTGTTT TTTATTCAGA TTGGGAAATC CGTTCTATTT TGTGAATTTG 1928 GGACATAATA ACAGCAGTGG AGTAAGTATT TAGAAGTGTG AATTCACCGT 1978 GAAATAGGTG AGATAAATTA AAAGATACTT AATTCCCGCC TTATGCCTCA 2028 GTCTATTCTG TAAAATTTAA AAATATATAT GCATACCTGG ATTTCCTTGG 2078 CTTCGTGAAT GTAAGAGAAA TTAAATCTGA ATAAATAATT CTTTCTGTTA 2128 ACTGGCTCAT TTCTTCTCTA TGCACTGAGC ATCTGCTCTG TGGAAGGCCC 2178 AGGATTAGTA GTGGAGATAC TAGGGTAAGC CAGACACACA CCTACCGATA 2228 GGGTATTAAG AGTCTAGGAG CGCGGTCATA TAATTAAGGT GACAAGATGT 2278 CCTCTAAGAT GTAGGGGAAA AGTAACGAGT GTGGGTATGG GGCTCCAGGT 2328 GAGAGTGGTC GGGTGTAAAT TCCCTGTGTG GGGCCTTTTG GGCTTTGGGA 2378 AACTGCATTT TCTTCTGAGG GATCTGATTC TAATGAAGCT TGGTGGGTCC 2428 AGGGCCAGAT TCTCAGAGGG AGAGGGAAAA GCCCAGATTG GAAAAGTTGC 2478 TCTGAGCAGT TCCTTTGTGA CAATGGATGA ACAGAGAGGA GCCTCTACCT 2528 GGG 2531 2531 base pairs nucleic acid single linear genomic DNA MAGE-41 gene 14 GGATCCAGGC CCTGCCTGGA GAAATGTGAG GGCCCTGAGT GAACACAGTG 50 GGGATCATCC ACTCCATGAG AGTGGGGACC TCACAGAGTC CAGCCTACCC 100 TCTTGATGGC ACTGAGGGAC CGGGGCTGTG CTTACAGTCT GCACCCTAAG 150 GGCCCATGGA TTCCTCTCCT AGGAGCTCCA GGAACAAGGC AGTGAGGCCT 200 TGGTCTGAGA CAGTGTCCTC AGGTTACAGA GCAGAGGATG CACAGGCTGT 250 GCCAGCAGTG AATGTTTGCC CTGAATGCAC ACCAAGGGCC CCACCTGCCA 300 CAAGACACAT AGGACTCCAA AGAGTCTGGC CTCACCTCCC TACCATCAAT 350 CCTGCAGAAT CGACCTCTGC TGGCCGGCTA TACCCTGAGG TGCTCTCTCA 400 CTTCCTCCTT CAGGTTCTGA GCAGACAGGC CAACCGGAGA CAGGATTCCC 450 TGGAGGCCAC AGAGGAGCAC CAAGGAGAAG ATCTGTAAGT AAGCCTTTGT 500 TAGAGCCTCT AAGATTTGGT TCTCAGCTGA GGTCTCTCAC ATGCTCCCTC 550 TCTCCGTAGG CCTGTGGGTC CCCATTGCCC AGCTTTTGCC TGCACTCTTG 600 CCTGCTGCCC TGAGCAGAGT CATC 624 ATG TCT TCT GAG CAG AAG AGT CAG CAC TGC AAG CCT GAG GAA 666 GGC GTT GAG GCC CAA GAA GAG GCC CTG GGC CTG GTG GGT GCG 708 CAG GCT CCT ACT ACT GAG GAG CAG GAG GCT GCT GTC TCC TCC 750 TCC TCT CCT CTG GTC CCT GGC ACC CTG GAG GAA GTG CCT GCT 792 GCT GAG TCA GCA GGT CCT CCC CAG AGT CCT CAG GGA GCC TCT 834 GCC TTA CCC ACT ACC ATC AGC TTC ACT TGC TGG AGG CAA CCC 876 AAT GAG GGT TCC AGC AGC CAA GAA GAG GAG GGG CCA AGC ACC 918 TCG CCT GAC GCA GAG TCC TTG TTC CGA GAA GCA CTC AGT AAC 960 AAG GTG GAT GAG TTG GCT CAT TTT CTG CTC CGC AAG TAT CGA 1002 GCC AAG GAG CTG GTC ACA AAG GCA GAA ATG CTG GAG AGA GTC 1044 ATC AAA AAT TAC AAG CGC TGC TTT CCT GTG ATC TTC GGC AAA 1086 GCC TCC GAG TCC CTG AAG ATG ATC TTT GGC ATT GAC GTG AAG 1128 GAA GTG GAC CCC ACC AGC AAC ACC TAC ACC CTT GTC ACC TGC 1170 CTG GGC CTT TCC TAT GAT GGC CTG CTG GGT AAT AAT CAG ATC 1212 TTT CCC AAG ACA GGC CTT CTG ATA ATC GTC CTG GGC ACA ATT 1254 GCA ATG GAG GGC GAC AGC GCC TCT GAG GAG GAA ATC TGG GAG 1296 GAG CTG GGT GTG ATG GGG GTG TAT GAT GGG AGG GAG CAC ACT 1338 GTC TAT GGG GAG CCC AGG AAA CTG CTC ACC CAA GAT TGG GTG 1380 CAG GAA AAC TAC CTG GAG TAC CGG CAG GTA CCC GGC AGT AAT 1422 CCT GCG CGC TAT GAG TTC CTG TGG GGT CCA AGG GCT CTG GCT 1464 GAA ACC AGC TAT GTG AAA GTC CTG GAG CAT GTG GTC AGG GTC 1506 AAT GCA AGA GTT CGC ATT GCC TAC CCA TCC CTG CGT GAA GCA 1548 GCT TTG TTA GAG GAG GAA GAG GGA GTC TGA 1578 GCATGAGTTG CAGCCAGGGC TGTGGGGAAG GGGCAGGGCT GGGCCAGTGC 1628 ATCTAACAGC CCTGTGCAGC AGCTTCCCTT GCCTCGTGTA ACATGAGGCC 1678 CATTCTTCAC TCTGTTTGAA GAAAATAGTC AGTGTTCTTA GTAGTGGGTT 1728 TCTATTTTGT TGGATGACTT GGAGATTTAT CTCTGTTTCC TTTTACAATT 1778 GTTGAAATGT TCCTTTTAAT GGATGGTTGA ATTAACTTCA GCATCCAAGT 1828 TTATGAATCG TAGTTAACGT ATATTGCTGT TAATATAGTT TAGGAGTAAG 1878 AGTCTTGTTT TTTATTCAGA TTGGGAAATC CGTTCTATTT TGTGAATTTG 1928 GGACATAATA ACAGCAGTGG AGTAAGTATT TAGAAGTGTG AATTCACCGT 1978 GAAATAGGTG AGATAAATTA AAAGATACTT AATTCCCGCC TTATGCCTCA 2028 GTCTATTCTG TAAAATTTAA AAATATATAT GCATACCTGG ATTTCCTTGG 2078 CTTCGTGAAT GTAAGAGAAA TTAAATCTGA ATAAATAATT CTTTCTGTTA 2128 ACTGGCTCAT TTCTTCTCTA TGCACTGAGC ATCTGCTCTG TGGAAGGCCC 2178 AGGATTAGTA GTGGAGATAC TAGGGTAAGC CAGACACACA CCTACCGATA 2228 GGGTATTAAG AGTCTAGGAG CGCGGTCATA TAATTAAGGT GACAAGATGT 2278 CCTCTAAGAT GTAGGGGAAA AGTAACGAGT GTGGGTATGG GGCTCCAGGT 2328 GAGAGTGGTC GGGTGTAAAT TCCCTGTGTG GGGCCTTTTG GGCTTTGGGA 2378 AACTCCATTT TCTTCTGAGG GATCTGATTC TAATGAAGCT TGGTGGGTCC 2428 AGGGCCAGAT TCTCAGAGGG AGAGGGAAAA GCCCAGATTG GAAAAGTTGC 2478 TCTGAGCGGT TCCTTTGTGA CAATGGATGA ACAGAGAGGA GCCTCTACCT 2528 GGG 2531 1068 base pairs nucleic acid single linear cDNA to mRNA cDNA MAGE-4 15 G GGG CCA AGC ACC TCG CCT GAC GCA GAG TCC TTG TTC CGA 40 GAA GCA CTC AGT AAC AAG GTG GAT GAG TTG GCT CAT TTT CTG 82 CTC CGC AAG TAT CGA GCC AAG GAG CTG GTC ACA AAG GCA GAA 124 ATG CTG GAG AGA GTC ATC AAA AAT TAC AAG CGC TGC TTT CCT 166 GTG ATC TTC GGC AAA GCC TCC GAG TCC CTG AAG ATG ATC TTT 208 GGC ATT GAC GTG AAG GAA GTG GAC CCC GCC AGC AAC ACC TAC 250 ACC CTT GTC ACC TGC CTG GGC CTT TCC TAT GAT GGC CTG CTG 292 GGT AAT AAT CAG ATC TTT CCC AAG ACA GGC CTT CTG ATA ATC 334 GTC CTG GGC ACA ATT GCA ATG GAG GGC GAC AGC GCC TCT GAG 376 GAG GAA ATC TGG GAG GAG CTG GGT GTG ATG GGG GTG TAT GAT 418 GGG AGG GAG CAC ACT GTC TAT GGG GAG CCC AGG AAA CTG CTC 460 ACC CAA GAT TGG GTG CAG GAA AAC TAC CTG GAG TAC CGG CAG 502 GTA CCC GGC AGT AAT CCT GCG CGC TAT GAG TTC CTG TGG GGT 544 CCA AGG GCT CTG GCT GAA ACC AGC TAT GTG AAA GTC CTG GAG 586 CAT GTG GTC AGG GTC AAT GCA AGA GTT CGC ATT GCC TAC CCA 628 TCC CTG CGT GAA GCA GCT TTG TTA GAG GAG GAA GAG GGA GTC 670 TGAGCATGAG TTGCAGCCAG GGCTGTGGGG AAGGGGCAGG GCTGGGCCAG 720 TGCATCTAAC AGCCCTGTGC AGCAGCTTCC CTTGCCTCGT GTAACATGAG 770 GCCCATTCTT CACTCTGTTT GAAGAAAATA GTCAGTGTTC TTAGTAGTGG 820 GTTTCTATTT TGTTGGATGA CTTGGAGATT TATCTCTGTT TCCTTTTACA 870 ATTGTTGAAA TGTTCCTTTT AATGGATGGT TGAATTAACT TCAGCATCCA 920 AGTTTATGAA TCGTAGTTAA CGTATATTGC TGTTAATATA GTTTAGGAGT 970 AAGAGTCTTG TTTTTTATTC AGATTGGGAA ATCCGTTCTA TTTTGTGAAT 1020 TTGGGACATA ATAACAGCAG TGGAGTAAGT ATTTAGAAGT GTGAATTC 1068 2226 base pairs nucleic acid single linear genomic DNA MAGE-5 gene 16 GGATCCAGGC CTTGCCAGGA GAAAGGTGAG GGCCCTGTGT GAGCACAGAG 50 GGGACCATTC ACCCCAAGAG GGTGGAGACC TCACAGATTC CAGCCTACCC 100 TCCTGTTAGC ACTGGGGGCC TGAGGCTGTG CTTGCAGTCT GCACCCTGAG 150 GGCCCATGCA TTCCTCTTCC AGGAGCTCCA GGAAACAGAC ACTGAGGCCT 200 TGGTCTGAGG CCGTGCCCTC AGGTCACAGA GCAGAGGAGA TGCAGACGTC 250 TAGTGCCAGC AGTGAACGTT TGCCTTGAAT GCACACTAAT GGCCCCCATC 300 GCCCCAGAAC ATATGGGACT CCAGAGCACC TGGCCTCACC CTCTCTACTG 350 TCAGTCCTGC AGAATCAGCC TCTGCTTGCT TGTGTACCCT GAGGTGCCCT 400 CTCACTTTTT CCTTCAGGTT CTCAGGGGAC AGGCTGACCA GGATCACCAG 450 GAAGCTCCAG AGGATCCCCA GGAGGCCCTA GAGGAGCACC AAAGGAGAAG 500 ATCTGTAAGT AAGCCTTTGT TAGAGCCTCC AAGGTTCAGT TTTTAGCTGA 550 GGCTTCTCAC ATGCTCCCTC TCTCTCCAGG CCAGTGGGTC TCCATTGCCC 600 AGCTCCTGCC CACACTCCTG CCTGTTGCGG TGACCAGAGT CGTC 644 ATG TCT CTT GAG CAG AAG AGT CAG CAC TGC AAG CCT GAG GAA 686 CTC CTC TGG TCC CAG GCA CCC TGG GGG AGG TGC CTG CTG CTG 728 GGT CAC CAG GTC CTC TCA AGA GTC CTC AGG GAG CCT CCG CCA 770 TCC CCA CTG CCA TCG ATT TCA CTC TAT GGA GGC AAT CCA TTA 812 AGG GCT CCA GCA ACC AAG AAG AGG AGG GGC CAA GCA CCT CCC 854 CTG ACC CAG AGT CTG TGT TCC GAG CAG CAC TCA GTA AGA AGG 896 TGG CTG ACT TGA 908 TTCATTTTCT GCTCCTCAAG TATTAAGTCA AGGAGCTGGT CACAAAGGCA 958 GAAATGCTGG AGAGCGTCAT CAAAAATTAC AAGCGCTGCT TTCCTGAGAT 1008 CTTCGGCAAA GCCTCCGAGT CCTTGCAGCT GGTCTTTGGC ATTGACGTGA 1058 AGGAAGCGGA CCCCACCAGC AACACCTACA CCCTTGTCAC CTGCCTGGGA 1108 CTCCTATGAT GGCCTGCTGG TTGATAATAA TCAGATCATG CCCAAGACGG 1158 GCCTCCTGAT AATCGTCTTG GGCATGATTG CAATGGAGGG CAAATGCGTC 1208 CCTGAGGAGA AAATCTGGGA GGAGCTGAGT GTGATGAAGG TGTATGTTGG 1258 GAGGGAGCAC AGTGTCTGTG GGGAGCCCAG GAAGCTGCTC ACCCAAGATT 1308 TGGTGCAGGA AAACTACCTG GAGTACCGGC AGGTGCCCAG CAGTGATCCC 1358 ATATGCTATG AGTTACTGTG GGGTCCAAGG GCACTCGCTG CTTGAAAGTA 1408 CTGGAGCACG TGGTCAGGGT CAATGCAAGA GTTCTCATTT CCTACCCATC 1458 CCTGCGTGAA GCAGCTTTGA GAGAGGAGGA AGAGGGAGTC TGAGCATGAG 1508 CTGCAGCCAG GGCCACTGCG AGGGGGGCTG GGCCAGTGCA CCTTCCAGGG 1558 CTCCGTCCAG TAGTTTCCCC TGCCTTAATG TGACATGAGG CCCATTCTTC 1608 TCTCTTTGAA GAGAGCAGTC AACATTCTTA GTAGTGGGTT TCTGTTCTAT 1658 TGGATGACTT TGAGATTTGT CTTTGTTTCC TTTTGGAATT GTTCAAATGT 1708 TTCTTTTAAT GGGTGGTTGA ATGAACTTCA GCATTCAAAT TTATGAATGA 1758 CAGTAGTCAC ACATAGTGCT GTTTATATAG TTTAGGAGTA AGAGTCTTGT 1808 TTTTTATTCA GATTGGGAAA TCCATTCCAT TTTGTGAATT GGGACATAGT 1858 TACAGCAGTG GAATAAGTAT TCATTTAGAA ATGTGAATGA GCAGTAAAAC 1908 TGATGACATA AAGAAATTAA AAGATATTTA ATTCTTGCTT ATACTCAGTC 1958 TATTCGGTAA AATTTTTTTT AAAAAATGTG CATACCTGGA TTTCCTTGGC 2008 TTCTTTGAGA ATGTAAGACA AATTAAATCT GAATAAATCA TTCTCCCTGT 2058 TCACTGGCTC ATTTATTCTC TATGCACTGA GCATTTGCTC TGTGGAAGGC 2108 CCTGGGTTAA TAGTGGAGAT GCTAAGGTAA GCCAGACTCA CCCCTACCCA 2158 CAGGGTAGTA AAGTCTAGGA GCAGCAGTCA TATAATTAAG GTGGAGAGAT 2208 GCCCTCTAAG ATGTAGAG 2226 2305 base pairs nucleic acid single linear genomic DNA MAGE-51 gene 17 GGATCCAGGC CTTGCCAGGA GAAAGGTGAG GGCCCTGTGT GAGCACAGAG 50 GGGACCATTC ACCCCAAGAG GGTGGAGACC TCACAGATTC CAGCCTACCC 100 TCCTGTTAGC ACTGGGGGCC TGAGGCTGTG CTTGCAGTCT GCACCCTGAG 150 GGCCCATGCA TTCCTCTTCC AGGAGCTCCA GGAAACAGAC ACTGAGGCCT 200 TGGTCTGAGG CCGTGCCCTC AGGTCACAGA GCAGAGGAGA TGCAGACGTC 250 TAGTGCCAGC AGTGAACGTT TGCCTTGAAT GCACACTAAT GGCCCCCATC 300 GCCCCAGAAC ATATGGGACT CCAGAGCACC TGGCCTCACC CTCTCTACTG 350 TCAGTCCTGC AGAATCAGCC TCTGCTTGCT TGTGTACCCT GAGGTGCCCT 400 CTCACTTTTT CCTTCAGGTT CTCAGGGGAC AGGCTGACCA GGATCACCAG 450 GAAGCTCCAG AGGATCCCCA GGAGGCCCTA GAGGAGCACC AAAGGAGAAG 500 ATCTGTAAGT AAGCCTTTGT TAGAGCCTCC AAGGTTCAGT TTTTAGCTGA 550 GGCTTCTCAC ATGCTCCCTC TCTCTCCAGG CCAGTGGGTC TCCATTGCCC 600 AGCTCCTGCC CACACTCCTG CCTGTTGCGG TGACCAGAGT CGTC 644 ATG TCT CTT GAG CAG AAG AGT CAG CAC TGC AAG CCT GAG GAA 686 GGC CTT GAC ACC CAA GAA GAG CCC TGG GCC TGG TGG GTG TGC 728 AGG CTG CCA CTA CTG AGG AGC AGG AGG CTG TGT CCT CCT CCT 770 CTC CTC TGG TCC CAG GCA CCC TGG GGG AGG TGC CTG CTG CTG 812 GGT CAC CAG GTC CTC TCA AGA GTC CTC AGG GAG CCT CCG CCA 854 TCC CCA CTG CCA TCG ATT TCA CTC TAT GGA GGC AAT CCA TTA 896 AGG GCT CCA GCA ACC AAG AAG AGG AGG GGC CAA GCA CCT CCC 938 CTG ACC CAG AGT CTG TGT TCC GAG CAG CAC TCA GTA AGA AGG 980 TGG CTG ACT TGA 992 TTCATTTTCT GCTCCTCAAG TATTAAGTCA AGGAGCCGGT CACAAAGGCA 1042 GAAATGCTGG AGAGCGTCAT CAAAAATTAC AAGCGCTGCT TTCCTGAGAT 1092 CTTCGGCAAA GCCTCCGAGT CCTTGCAGCT GGTCTTTGGC ATTGACGTGA 1142 AGGAAGCGGA CCCCACCAGC AACACCTACA CCCTTGTCAC CTGCCTGGGA 1192 CTCCTATGAT GGCCTGGTGG TTTAATCAGA TCATGCCCAA GACGGGCCTC 1242 CTGATAATCG TCTTGGGCAT GATTGCAATG GAGGGCAAAT GCGTCCCTGA 1292 GGAGAAAATC TGGGAGGAGC TGGGTGTGAT GAAGGTGTAT GTTGGGAGGG 1342 AGCACAGTGT CTGTGGGGAG CCCAGGAAGC TGCTCACCCA AGATTTGGTG 1392 CAGGAAAACT ACCTGGAGTA CCGCAGGTGC CCAGCAGTGA TCCCATATGC 1442 TATGAGTTAC TGTGGGGTCC AAGGGCACTC GCTGCTTGAA AGTACTGGAG 1492 CACGTGGTCA GGGTCAATGC AAGAGTTCTC ATTTCCTACC CATCCCTGCA 1542 TGAAGCAGCT TTGAGAGAGG AGGAAGAGGG AGTCTGAGCA TGAGCTGCAG 1592 CCAGGGCCAC TGCGAGGGGG GCTGGGCCAG TGCACCTTCC AGGGCTCCGT 1642 CCAGTAGTTT CCCCTGCCTT AATGTGACAT GAGGCCCATT CTTCTCTCTT 1692 TGAAGAGAGC AGTCAACATT CTTAGTAGTG GGTTTCTGTT CTATTGGATG 1742 ACTTTGAGAT TTGTCTTTGT TTCCTTTTGG AATTGTTCAA ATGTTCCTTT 1792 TAATGGGTGG TTGAATGAAC TTCAGCATTC AAATTTATGA ATGACAGTAG 1842 TCACACATAG TGCTGTTTAT ATAGTTTAGG AGTAAGAGTC TTGTTTTTTA 1892 TTCAGATTGG GAAATCCATT CCATTTTGTG AATTGGGACA TAGTTACAGC 1942 AGTGGAATAA GTATTCATTT AGAAATGTGA ATGAGCAGTA AAACTGATGA 1992 GATAAAGAAA TTAAAAGATA TTTAATTCTT GCCTTATACT CAGTCTATTC 2042 GGTAAAATTT TTTTTTAAAA ATGTGCATAC CTGGATTTCC TTGGCTTCTT 2092 TGAGAATGTA AGACAAATTA AATCTGAATA AATCATTCTC CCTGTTCACT 2142 GGCTCATTTA TTCTCTATGC ACTGAGCATT TGCTCTGTGG AAGGCCCTGG 2192 GTTAATAGTG GAGATGCTAA GGTAAGCCAG ACTCACCCCT ACCCACAGGG 2242 TAGTAAAGTC TAGGAGCAGC AGTCATATAA TTAAGGTGGA GAGATGCCCT 2292 CTAAGATGTA GAG 2305 225 base pairs nucleic acid single linear cDNA MAGE-6 gene 18 TAT TTC TTT CCT GTG ATC TTC AGC AAA GCT TCC GAT TCC TTG 42 CAG CTG GTC TTT GGC ATC GAG CTG ATG GAA GTG GAC CCC ATC 84 GGC CAC GTG TAC ATC TTT GCC ACC TGC CTG GGC CTC TCC TAC 126 GAT GGC CTG CTG GGT GAC AAT CAG ATC ATG CCC AGG ACA GGC 168 TTC CTG ATA ATC ATC CTG GCC ATA ATC GCA AGA GAG GGC GAC 210 TGT GCC CCT GAG GAG 225 1947 base pairs nucleic acid single linear genomic DNA MAGE-7 gene 19 TGAATGGACA ACAAGGGCCC CACACTCCCC AGAACACAAG GGACTCCAGA 50 GAGCCCAGCC TCACCTTCCC TACTGTCAGT CCTGCAGCCT CAGCCTCTGC 100 TGGCCGGCTG TACCCTGAGG TGCCCTCTCA CTTCCTCCTT CAGGTTCTCA 150 GCGGACAGGC CGGCCAGGAG GTCAGAAGCC CCAGGAGGCC CCAGAGGAGC 200 ACCGAAGGAG AAGATCTGTA AGTAGGCCTT TGTTAGGGCC TCCAGGGCGT 250 GGTTCACAAA TGAGGCCCCT CACAAGCTCC TTCTCTCCCC AGATCTGTGG 300 GTTCCTCCCC ATCGCCCAGC TGCTGCCCGC ACTCCAGCCT GCTGCCCTGA 350 CCAGAGTCAT CATGTCTTCT GAGCAGAGGA GTCAGCACTG CAAGCCTGAG 400 GATGCCTTGA GGCCCAAGGA CAGGAGGCTC TGGGCCTGGT GGGTGCGCAG 450 GCTCCCGCCA CCGAGGAGCA CGAGGCTGCC TCCTCCTTCA CTCTGATTGA 500 AGGCACCCTG GAGGAGGTGC CTGCTGCTGG GTCCCCCAGT CCTCCCCTGA 550 GTCTCAGGGT TCCTCCTTTT CCCTGACCAT CAGCAACAAC ACTCTATGGA 600 GCCAATCCAG TGAGGGCACC AGCAGCCGGG AAGAGGAGGG GCCAACCACC 650 TAGACACACC CCGCTCACCT GGCGTCCTTG TTCCA 685 ATG GGA AGG TGG CTG AGT TGG TTC GCT TCC TGC TGC ACA AGT 727 ATC GAG TCA AGG AGC TGG TCA CAA AGG CAG AAA TGC TGG ACA 769 GTG TCA TCA AAA ATT ACA AGC ACT AGT TTC CTT GTG ATC TAT 811 GGC AAA GCC TCA GAG TGC ATG CAG GTG ATG TTT GGC ATT GAC 853 ATG AAG GAA GTG GAC CCC GCG GCC ACT CCT ACG TCC TTG TCA 895 CCT GCT TGG GCC TCT CCT ACA ATG GCC TGC TGG GTG ATG ATC 937 AGA GCA TGC CCG AGA CCG GCC TTC TGA 964 TTATGGTCTT GACCATGATC TTAATGGAGG GCCACTGTGC CCCTGAGGAG 1014 GCAATCTGGG AAGCGTTGAG TGTAATGGTG TATGATGGGA TGGAGCAGTT 1064 TCTTTGGGCA GCTGAGGAAG CTGCTCACCC AAGATTGGGT GCAGGAAAAC 1114 TACCTGCAAT ACCGCCAGGT GCCCAGCAGT GATCCCCCGT GCTACCAGTT 1164 CCTGTGGGGT CCAAGGGCCC TCATTGAAAC CAGCTATGTG AAAGTCCTGG 1214 AGTATGCAGC CAGGGTCAGT ACTAAAGAGA GCATTTCCTA CCCATCCCTG 1264 CATGAAGAGG CTTTGGGAGA GGAGGAAGAG GGAGTCTGAG CAGAAGTTGC 1314 AGCCAGGGCC AGTGGGGCAG ATTGGGGGAG GGCCTGGGCA GTGCACGTTC 1364 CACACATCCA CCACCTTCCC TGTCCTGTTA CATGAGGCCC ATTCTTCACT 1414 CTGTGTTTGA AGAGAGCAGT CAATGTTCTC AGTAGCGGGG AGTGTGTTGG 1464 GTGTGAGGGA ATACAAGGTG GACCATCTCT CAGTTCCTGT TCTCTTGGGC 1514 GATTTGGAGG TTTATCTTTG TTTCCTTTTG CAGTCGTTCA AATGTTCCTT 1564 TTAATGGATG GTGTAATGAA CTTCAACATT CATTTCATGT ATGACAGTAG 1614 GCAGACTTAC TGTTTTTTAT ATAGTTAAAA GTAAGTGCAT TGTTTTTTAT 1664 TTATGTAAGA AAATCTATGT TATTTCTTGA ATTGGGACAA CATAACATAG 1714 CAGAGGATTA AGTACCTTTT ATAATGTGAA AGAACAAAGC GGTAAAATGG 1764 GTGAGATAAA GAAATAAAGA AATTAAATTG GCTGGGCACG GTGGCTCACG 1814 CCTGTAATCC CAGCACTTTA GGAGGCAGAG GCACGGGGAT CACGAGGTCA 1864 GGAGATCGAG ACCATTCTGG CTAACACAGT GAAACACCAT CTCTATTAAA 1914 AATACAAAAC TTAGCCGGGC GTGGTGGCGG GTG 1947 1810 base pairs nucleic acid single linear genomic DNA MAGE-8 gene 20 GAGCTCCAGG AACCAGGCTG TGAGGTCTTG GTCTGAGGCA GTATCTTCAA 50 TCACAGAGCA TAAGAGGCCC AGGCAGTAGT AGCAGTCAAG CTGAGGTGGT 100 GTTTCCCCTG TATGTATACC AGAGGCCCCT CTGGCATCAG AACAGCAGGA 150 ACCCCACAGT TCCTGGCCCT ACCAGCCCTT TTGTCAGTCC TGGAGCCTTG 200 GCCTTTGCCA GGAGGCTGCA CCCTGAGATG CCCTCTCAAT TTCTCCTTCA 250 GGTTCGCAGA GAACAGGCCA GCCAGGAGGT CAGGAGGCCC CAGAGAAGCA 300 CTGAAGAAGA CCTGTAAGTA GACCTTTGTT AGGGCATCCA GGGTGTAGTA 350 CCCAGCTGAG GCCTCTCACA CGCTTCCTCT CTCCCCAGGC CTGTGGGTCT 400 CAATTGCCCA GCTCCGGCCC ACACTCTCCT GCTGCCCTGA CCTGAGTCAT 450 C 451 ATG CTT CTT GGG CAG AAG AGT CAG CGC TAC AAG GCT GAG GAA 493 GGC CTT CAG GCC CAA GGA GAG GCA CCA GGG CTT ATG GAT GTG 535 CAG ATT CCC ACA GCT GAG GAG CAG AAG GCT GCA TCC TCC TCC 577 TCT ACT CTG ATC ATG GGA ACC CTT GAG GAG GTG ACT GAT TCT 619 GGG TCA CCA AGT CCT CCC CAG AGT CCT GAG GGT GCC TCC TCT 661 TCC CTG ACT GTC ACC GAC AGC ACT CTG TGG AGC CAA TCC GAT 703 GAG GGT TCC AGC AGC AAT GAA GAG GAG GGG CCA AGC ACC TCC 745 CCG GAC CCA GCT CAC CTG GAG TCC CTG TTC CGG GAA GCA CTT 787 GAT GAG AAA GTG GCT GAG TTA GTT CGT TTC CTG CTC CGC AAA 829 TAT CAA ATT AAG GAG CCG GTC ACA AAG GCA GAA ATG CTT GAG 871 AGT GTC ATC AAA AAT TAC AAG AAC CAC TTT CCT GAT ATC TTC 913 AGC AAA GCC TCT GAG TGC ATG CAG GTG ATC TTT GGC ATT GAT 955 GTG AAG GAA GTG GAC CCT GCC GGC CAC TCC TAC ATC CTT GTC 997 ACC TGC CTG GGC CTC TCC TAT GAT GGC CTG CTG GGT GAT GAT 1039 CAG AGT ACG CCC AAG ACC GGC CTC CTG ATA ATC GTC CTG GGC 1081 ATG ATC TTA ATG GAG GGC AGC CGC GCC CCG GAG GAG GCA ATC 1123 TGG GAA GCA TTG AGT GTG ATG GGG GCT GTA TGA 1156 TGGGAGGGAG CACAGTGTCT ATTGGAAGCT CAGGAAGCTG CTCACCCAAG 1206 AGTGGGTGCA GGAGAACTAC CTGGAGTACC GCCAGGCGCC CGGCAGTGAT 1256 CCTGTGCGCT ACGAGTTCCT GTGGGGTCCA AGGGCCCTTG CTGAAACCAG 1306 CTATGTGAAA GTCCTGGAGC ATGTGGTCAG GGTCAATGCA AGAGTTCGCA 1356 TTTCCTACCC ATCCCTGCAT GAAGAGGCTT TGGGAGAGGA GAAAGGAGTT 1406 TGAGCAGGAG TTGCAGCTAG GGCCAGTGGG GCAGGTTGTG GGAGGGCCTG 1456 GGCCAGTGCA CGTTCCAGGG CCACATCCAC CACTTTCCCT GCTCTGTTAC 1506 ATGAGGCCCA TTCTTCACTC TGTGTTTGAA GAGAGCAGTC ACAGTTCTCA 1556 GTAGTGGGGA GCATGTTGGG TGTGAGGGAA CACAGTGTGG ACCATCTCTC 1606 AGTTCCTGTT CTATTGGGCG ATTTGGAGGT TTATCTTTGT TTCCTTTTGG 1656 AATTGTTCCA ATGTTCCTTC TAATGGATGG TGTAATGAAC TTCAACATTC 1706 ATTTTATGTA TGACAGTAGA CAGACTTACT GCTTTTTATA TAGTTTAGGA 1756 GTAAGAGTCT TGCTTTTCAT TTATACTGGG AAACCCATGT TATTTCTTGA 1806 ATTC 1810 1412 base pairs nucleic acid single linear genomic DNA MAGE-9 gene 21 TCTGAGACAG TGTCCTCAGG TCGCAGAGCA GAGGAGACCC AGGCAGTGTC 50 AGCAGTGAAG GTGAAGTGTT CACCCTGAAT GTGCACCAAG GGCCCCACCT 100 GCCCCAGCAC ACATGGGACC CCATAGCACC TGGCCCCATT CCCCCTACTG 150 TCACTCATAG AGCCTTGATC TCTGCAGGCT AGCTGCACGC TGAGTAGCCC 200 TCTCACTTCC TCCCTCAGGT TCTCGGGACA GGCTAACCAG GAGGACAGGA 250 GCCCCAAGAG GCCCCAGAGC AGCACTGACG AAGACCTGTA AGTCAGCCTT 300 TGTTAGAACC TCCAAGGTTC GGTTCTCAGC TGAAGTCTCT CACACACTCC 350 CTCTCTCCCC AGGCCTGTGG GTCTCCATCG CCCAGCTCCT GCCCACGCTC 400 CTGACTGCTG CCCTGACCAG AGTCATC 427 ATG TCT CTC GAG CAG AGG AGT CCG CAC TGC AAG CCT GAT GAA 469 GAC CTT GAA GCC CAA GGA GAG GAC TTG GGC CTG ATG GGT GCA 511 CAG GAA CCC ACA GGC GAG GAG GAG GAG ACT ACC TCC TCC TCT 553 GAC AGC AAG GAG GAG GAG GTG TCT GCT GCT GGG TCA TCA AGT 595 CCT CCC CAG AGT CCT CAG GGA GGC GCT TCC TCC TCC ATT TCC 637 GTC TAC TAC ACT TTA TGG AGC CAA TTC GAT GAG GGC TCC AGC 679 AGT CAA GAA GAG GAA GAG CCA AGC TCC TCG GTC GAC CCA GCT 721 CAG CTG GAG TTC ATG TTC CAA GAA GCA CTG AAA TTG AAG GTG 763 GCT GAG TTG GTT CAT TTC CTG CTC CAC AAA TAT CGA GTC AAG 805 GAG CCG GTC ACA AAG GCA GAA ATG CTG GAG AGC GTC ATC AAA 847 AAT TAC AAG CGC TAC TTT CCT GTG ATC TTC GGC AAA GCC TCC 889 GAG TTC ATG CAG GTG ATC TTT GGC ACT GAT GTG AAG GAG GTG 931 GAC CCC GCC GGC CAC TCC TAC ATC CTT GTC ACT GCT CTT GGC 973 CTC TCG TGC GAT AGC ATG CTG GGT GAT GGT CAT AGC ATG CCC 1015 AAG GCC GCC CTC CTG ATC ATT GTC CTG GGT GTG ATC CTA ACC 1057 AAA GAC AAC TGC GCC CCT GAA GAG GTT ATC TGG GAA GCG TTG 1099 AGT GTG ATG GGG GTG TAT GTT GGG AAG GAG CAC ATG TTC TAC 1141 GGG GAG CCC AGG AAG CTG CTC ACC CAA GAT TGG GTG CAG GAA 1183 AAC TAC CTG GAG TAC CGG CAG GTG CCC GGC AGT GAT CCT GCG 1225 CAC TAC GAG TTC CTG TGG GGT TCC AAG GCC CAC GCT GAA ACC 1267 AGC TAT GAG AAG GTC ATA AAT TAT TTG GTC ATG CTC AAT GCA 1309 AGA GAG CCC ATC TGC TAC CCA TCC CTT TAT GAA GAG GTT TTG 1351 GGA GAG GAG CAA GAG GGA GTC TGA 1375 GCACCAGCCG CAGCCGGGGC CAAAGTTTGT GGGGTCA 1412 920 base pairs nucleic acid single linear genomic DNA MAGE-10 gene 22 ACCTGCTCCA GGACAAAGTG GACCCCACTG CATCAGCTCC ACCTACCCTA 50 CTGTCAGTCC TGGAGCCTTG GCCTCTGCCG GCTGCATCCT GAGGAGCCAT 100 CTCTCACTTC CTTCTTCAGG TTCTCAGGGG ACAGGGAGAG CAAGAGGTCA 150 AGAGCTGTGG GACACCACAG AGCAGCACTG AAGGAGAAGA CCTGTAAGTT 200 GGCCTTTGTT AGAACCTCCA GGGTGTGGTT CTCAGCTGTG GCCACTTACA 250 CCCTCCCTCT CTCCCCAGGC CTGTGGGTCC CCATCGCCCA AGTCCTGCCC 300 ACACTCCCAC CTGCTACCCT GATCAGAGTC ATC 333 ATG CCT CGA GCT CCA AAG CGT CAG CGC TGC ATG CCT GAA GAA 375 GAT CTT CAA TCC CAA AGT GAG ACA CAG GGC CTC GAG GGT GCA 417 CAG GCT CCC CTG GCT GTG GAG GAG GAT GCT TCA TCA TCC ACT 459 TCC ACC AGC TCC TCT TTT CCA TCC TCT TTT CCC TCC TCC TCC 501 TCT TCC TCC TCC TCC TCC TGC TAT CCT CTA ATA CCA AGC ACC 543 CCA GAG GAG GTT TCT GCT GAT GAT GAG ACA CCA AAT CCT CCC 585 CAG AGT GCT CAG ATA GCC TGC TCC TCC CCC TCG GTC GTT GCT 627 TCC CTT CCA TTA GAT CAA TCT GAT GAG GGC TCC AGC AGC CAA 669 AAG GAG GAG AGT CCA AGC ACC CTA CAG GTC CTG CCA GAC AGT 711 GAG TCT TTA CCC AGA AGT GAG ATA GAT GAA AAG GTG ACT GAT 753 TTG GTG CAG TTT CTG CTC TTC AAG TAT CAA ATG AAG GAG CCG 795 ATC ACA AAG GCA GAA ATA CTG GAG AGT GTC ATA AAA AAT TAT 837 GAA GAC CAC TTC CCT TTG TTG TTT AGT GAA GCC TCC GAG TGC 879 ATG CTG CTG GTC TTT GGC ATT GAT GTA AAG GAA GTG GAT CC 920 1107 base pairs nucleic acid single linear genomic DNA MAGE-11 gene 23 AGAGAACAGG CCAACCTGGA GGACAGGAGT CCCAGGAGAA CCCAGAGGAT 50 CACTGGAGGA GAACAAGTGT AAGTAGGCCT TTGTTAGATT CTCCATGGTT 100 CATATCTCAT CTGAGTCTGT TCTCACGCTC CCTCTCTCCC CAGGCTGTGG 150 GGCCCCATCA CCCAGATATT TCCCACAGTT CGGCCTGCTG ACCTAACCAG 200 AGTCATCATG CCTCTTGAGC AAAGAAGTCA GCACTGCAAG CCTGAGGAAG 250 CCTTCAGGCC CAAGAAGAAG ACCTGGGCCT GGTGGGTGCA CAGGCTCTCC 300 AAGCTGAGGA GCAGGAGGCT GCCTTCTTCT CCTCTACTCT GAATGTGGGC 350 ACTCTAGAGG AGTTGCCTGC TGCTGAGTCA CCAAGTCCTC CCCAGAGTCC 400 TCAGGAAGAG TCCTTCTCTC CCACTGCCAT GGATGCCATC TTTGGGAGCC 450 TATCTGATGA GGGCTCTGGC AGCCAAGAAA AGGAGGGGCC AAGTACCTCG 500 CCTGACCTGA TAGACCCTGA GTCCTTTTCC CAAGATATAC TACATGACAA 550 GATAATTGAT TTGGTTCATT TATTCTCCGC AAGTATCGAG TCAAGGGGCT 600 GGATCACAAAG GCAGAA 616 ATG CTG GGG AGT GTC ATC AAA AAT TAT GAG GAC TAC TTT CCT 658 GAG ATA TTT AGG GAA GCC TCT GTA TGC ATG CAA CTG CTC TTT 700 GGC ATT GAT GTG AAG GAA GTG GAC CCC ACT AGC CAC TCC TAT 742 GTC CTT GTC ACC TCC CTC AAC CTC TCT TAT GAT GGC ATA CAG 784 TGT AAT GAG CAG AGC ATG CCC AAG TCT GGC CTC CTG ATA ATA 826 GTC CTG GGT GTA ATC TTC ATG GAG GGG AAC TGC ATC CCT GAA 868 GAG GTT ATG TGG GAA GTC CTG AGC ATT ATG GGG GTG TAT GCT 910 GGA AGG GAG CAC TTC CTC TTT GGG GAG CCC AAG AGG CTC CTT 952 ACC CAA AAT TGG GTG CAG GAA AAG TAC CTG GTG TAC CGG CAG 994 GTG CCC GGC ACT GAT CCT GCA TGC TAT GAG TTC CTG TGG GGT 1036 CCA AGG GCC CAC GCT GAG ACC AGC AAG ATG AAA GTT CTT GAG 1078 TAC ATA GCC AAT GCC AAT GGG AGG GAT CC 1107 2150 base pairs nucleic acid single linear genomic DNA smage-I 24 TCTGTCTGCA TATGCCTCCA CTTGTGTGTA GCAGTCTCAA ATGGATCTCT 50 CTCTACAGAC CTCTGTCTGT GTCTGGCACC CTAAGTGGCT TTGCATGGGC 100 ACAGGTTTCT GCCCCTGCAT GGAGCTTAAA TAGATCTTTC TCCACAGGCC 150 TATACCCCTG CATTGTAAGT TTAAGTGGCT TTATGTGGAT ACAGGTCTCT 200 GCCCTTGTAT GCAGGCCTAA GTTTTTCTGT CTGCTTAACC CCTCCAAGTG 250 AAGCTAGTGA AAGATCTAAC CCACTTTTGG AAGTCTGAAA CTAGACTTTT 300 ATGCAGTGGC CTAACAAGTT TTAATTTCTT CCACAGGGTT TGCAGAAAAG 350 AGCTTGATCC ACGAGTTCAG AAGTCCTGGT ATGTTCCTAG AAAG 394 ATG TTC TCC TGG AAA GCT TCA AAA GCC AGG TCT CCA TTA AGT 436 CCA AGG TAT TCT CTA CCT GGT AGT ACA GAG GTA CTT ACA GGT 478 TGT CAT TCT TAT CCT TCC AGA TTC CTG TCT GCC AGC TCT TTT 520 ACT TCA GCC CTG AGC ACA GTC AAC ATG CCT AGG GGT CAA AAG 562 AGT AAG ACC CGC TCC CGT GCA AAA CGA CAG CAG TCA CGC AGG 604 GAG GTT CCA GTA GTT CAG CCC ACT GCA GAG GAA GCA GGG TCT 646 TCT CCT GTT GAC CAG AGT GCT GGG TCC AGC TTC CCT GGT GGT 688 TCT GCT CCT CAG GGT GTG AAA ACC CCT GGA TCT TTT GGT GCA 730 GGT GTA TCC TGC ACA GGC TCT GGT ATA GGT GGT AGA AAT GCT 772 GCT GTC CTG CCT GAT ACA AAA AGT TCA GAT GGC ACC CAG GCA 814 GGG ACT TCC ATT CAG CAC ACA CTG AAA GAT CCT ATC ATG AGG 856 AAG GCT AGT GTG CTG ATA GAA TTC CTG CTA GAT AAA TTT AAG 898 ATG AAA GAA GCA GTT ACA AGG AGT GAA ATG CTG GCA GTA GTT 940 AAC AAG AAG TAT AAG GAG CAA TTC CCT GAG ATC CTC AGG AGA 982 ACT TCT GCA CGC CTA GAA TTA GTC TTT GGT CTT GAG TTG AAG 1024 GAA ATT GAT CCC AGC ACT CAT TCC TAT TTG CTG GTA GGC AAA 1066 CTG GGT CTT TCC ACT GAG GGA AGT TTG AGT AGT AAC TGG GGG 1108 TTG CCT AGG ACA GGT CTC CTA ATG TCT GTC CTA GGT GTG ATC 1150 TTC ATG AAG GGT AAC CGT GCC ACT GAG CAA GAG GTC TGG CAA 1192 TTT CTG CAT GGA GTG GGG GTA TAT GCT GGG AAG AAG CAC TTG 1234 ATC TTT GGC GAG CCT GAG GAG TTT ATA AGA GAT GTA GTG CGG 1276 GAA AAT TAC CTG GAG TAC CGC CAG GTA CCT GGC AGT GAT CCC 1318 CCA AGC TAT GAG TTC CTG TGG GGA CCC AGA GCC CAT GCT GAA 1360 ACA ACC AAG ATG AAA GTC CTG GAA GTT TTA GCT AAA GTC AAT 1402 GGC ACA GTC CCT AGT GCC TTC CCT AAT CTC TAC CAG TTG GCT 1444 CTT AGA GAT CAG GCA GGA GGG GTG CCA AGA AGG AGA GTT CAA 1486 GGC AAG GGT GTT CAT TCC AAG GCC CCA TCC CAA AAG TCC TCT 1528 AAC ATG TAG 1537 TTGAGTCTGT TCTGTTGTGT TTGAAAAACA GTCAGGCTCC TAATCAGTAG 1587 AGAGTTCATA GCCTACCAGA ACCAACATGC ATCCATTCTT GGCCTGTTAT 1637 ACATTAGTAG AATGGAGGCT ATTTTTGTTA CTTTTCAAAT GTTTGTTTAA 1687 CTAAACAGTG CTTTTTGCCA TGCTTCTTGT TAACTGCATA AAGAGGTAAC 1737 TGTCACTTGT CAGATTAGGA CTTGTTTTGT TATTTGCAAC AAACTGGAAA 1787 ACATTATTTT GTTTTTACTA AAACATTGTG TAACATTGCA TTGGAGAAGG 1837 GATTGTCATG GCAATGTGAT ATCATACAGT GGTGAAACAA CAGTGAAGTG 1887 GGAAAGTTTA TATTGTTAAT TTTGAAAATT TTATGAGTGT GATTGCTGTA 1937 TACTTTTTTC TTTTTTGTAT AATGCTAAGT GAAATAAAGT TGGATTTGAT 1987 GACTTTACTC AAATTCATTA GAAAGTAAAT CGTAAAACTC TATTACTTTA 2037 TTATTTTCTT CAATTATGAA TTAAGCATTG GTTATCTGGA AGTTTCTCCA 2087 GTAGCACAGG ATCTAGTATG AAATGTATCT AGTATAGGCA CTGACAGTGA 2137 GTTATCAGAG TCT 2150 2099 base pairs nucleic acid single linear genomic DNA smage-II 25 ACCTTATTGG GTCTGTCTGC ATATGCCTCC ACTTGTGTGT AGCAGTCTCA 50 AATGGATCTC TCTCTACAGA CCTCTGTCTG TGTCTGGCAC CCTAAGTGGC 100 TTTGCATGGG CACAGGTTTC TGCCCCTGCA TGGAGCTTAA ATAGATCTTT 150 CTCCACAGGC CTATACCCCT GCATTGTAAG TTTAAGTGGC TTTATGTGGA 200 TACAGGTCTC TGCCCTTGTA TGCAGGCCTA AGTTTTTCTG TCTGCTTAGC 250 CCCTCCAAGT GAAGCTAGTG AAAGATCTAA CCCACTTTTG GAAGTCTGAA 300 ACTAGACTTT TATGCAGTGG CCTAACAAGT TTTAATTTCT TCCACAGGGT 350 TTGCAGAAAA GAGCTTGATC CACGAGTTCG GAAGTCCTGG TATGTTCCTA 400 GAAAGATGTT CTCCTGGAAA GCTTCAAAAG CCAGGTCTCC ATTAAGTCCA 450 AGGTATTCTC TACCTGGTAG TACAGAGGTA CTTACAGGTT GTCATTCTTA 500 TCTTTCCAGA TTCCTGTCTG CCAGCTCTTT TACTTCAGCC CTGAGCACAG 550 TCAACATGCC TAGGGGTCAA AAGAGTAAGA CCCGCTCCCG TGCAAAACGA 600 CAGCAGTCAC GCAGGGAGGT TCCAGTAGTT CAGCCCACTG CAGAGGAAGC 650 AGGGTCTTCT CCTGTTGACC AGAGTGCTGG GTCCAGCTTC CCTGGTGGTT 700 CTGCTCCTCA GGGTGTGAAA ACCCCTGGAT CTTTTGGTGC AGGTGTATCC 750 TGCACAGGCT CTGGTATAGG TGGTAGAAAT GCTGCTGTCC TGCCTGATAC 800 AAAAAGTTCA GATGGCACCC AGGCAGGGAC TTCCATTCAG CACACACTGA 850 AAGATCCTAT CATGAGGAAG GCTAGTGTGC TGATAGAATT CCTGCTAGAT 900 AAGTTTAAGA TGAAAGAAGC AGTTACAAGG AGTGAAATGC TGGCAGTAGT 950 TAACAAGAAG TATAAGGAGC AATTCCCTGA GATCCTCAGG AGAACTTCTG 1000 CACGCCTAGA ATTAGTCTTT GGTCTTGAGT TGAAGGAAAT TGATCCCAGC 1050 ACTCATTCCT ATTTGCTGGT AGGCAAACTG GGTCTTTCCA CTGAGGGAAG 1100 TTTGAGTAGT AACTGGGGGT TGCCTAGGAC AGGTCTCCTA ATGTCTGTCC 1150 TAGGTGTGAT CTTCATGAAG GGTAACCGTG CCACTGAGCA AGAGGTCTGG 1200 CAATTTCTGC ATGGAGTGGG GGTATATGCT GGGAAGAAGC ACTTGATCTT 1250 TGGCGAGCCT GAGGAGTTTA TAAGAGATGT AGTGCGGGAA AATTACCTGG 1300 AGTACCGCCA GGTACCTGGC AGTGATCCCC CAAGCTATGA GTTCCTGTGG 1350 GGACCCAGAG CCCATGCTGA AACAACCAAG ATGAAAGTCC TGGAAGTTTT 1400 AGCTAAAGTC AATGGCACAG TCCCTAGTGC CTTCCCTAAT CTCTACCAGT 1450 TGGCTCTTAG AGATCAGGCA GGAGGGGTGC CAAGAAGGAG AGTTCAAGGC 1500 AAGGGTGTTC ATTCCAAGGC CCCATCCCAA AAGTCCTCTA ACATGTAGTT 1550 GAGTCTGTTC TGTTGTGTTT GAAAAACAGT CAGGCTCCTA ATCAGTAGAG 1600 AGTTCATAGC CTACCAGAAC CAACATGCAT CCATTCTTGG CCTGTTATAC 1650 ATTAGTAGAA TGGAGGCTAT TTTTGTTACT TTTCAAATGT TTGTTTAACT 1700 AAACAGTGCT TTTTGCCATG CTTCTTGTTA ACTGCATAAA GAGGTAACTG 1750 TCACTTGTCA GATTAGGACT TGTTTTGTTA TTTGCAACAA ACTGGAAAAC 1800 ATTATTTTGT TTTTACTAAA ACATTGTGTA ACATTGCATT GGAGAAGGGA 1850 TTGTCATGGC AATGTGATAT CATACAGTGG TGAAACAACA GTGAAGTGGG 1900 AAAGTTTATA TTGTTAGTTT TGAAAATTTT ATGAGTGTGA TTGCTGTATA 1950 CTTTTTTCTT TTTTGTATAA TGCTAAGTGA AATAAAGTTG GATTTGATGA 2000 CTTTACTCAA ATTCATTAGA AAGTAAATCA TAAAACTCTA TTACTTTATT 2050 ATTTTCTTCA ATTATTAATT AAGCATTGGT TATCTGGAAG TTTCTCCAG 2099 9 amino acids amino acids linear protein 26 Glu Ala Asp Pro Thr Gly His Ser Tyr 5 20 nucleotides nucleic acid single linear 27 ACTCAGCTCC TCCCAGATTT 20 17 nucleotides nucleic acid single linear 28 GAAGAGGAGG GGCCAAG 17 18 nucleotides nucleic acid single linear 29 TCTTGTATCC TGGAGTCC 18 18 nucleotides nucleic acid single linear 30 TTGCCAAGAT CTCAGGAA 18 

We claim:
 1. An isolated nucleic acid molecule which encodes a tumor rejection antigen precursor expressed in melanoma cells, wherein the complementary sequence of said isolated nucleic acid molecule hybridizes to the nucleotide sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 10 at 0.1×SSC, 0.1% SDS for 30 minutes at 65° C.
 2. The isolated nucleic acid molecule of claim 1, wherein said isolated nucleic acid molecule is genomic DNA.
 3. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid molecule is cDNA.
 4. The isolated nucleic acid molecule of claim 1, comprising SEQ ID NO:
 10. 5. An expression vector comprising the isolated nucleic acid molecule of claim 1, operably linked to a promoter.
 6. A host cell transformed or transfected with the isolated nucleic acid molecule of claim
 1. 7. The host cell of claim 6, wherein said cell is a fibroblast.
 8. The host cell of claim 6, wherein said cell is a mammalian cell.
 9. An isolated nucleic acid molecule which encodes a fragment of a tumor rejection antigen precursor expressed in melanoma cells, wherein the complementary sequence of said isolated nucleic acid molecule hybridizes to the nucleotide sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 10 at 0.1×SSC, 0.1% SDS for 30 minutes at 65° C.
 10. The isolated nucleic acid molecule of claim 9, wherein said isolated nucleic acid molecule is genomic DNA.
 11. An expression vector comprising the isolated nucleic acid molecule of claim 9, operably linked to a promoter.
 12. A host cell transformed or transfected with the isolated nucleic acid molecule of claim
 9. 13. The host cell of claim 12, wherein said cell is a fibroblast.
 14. The host cell of claim 12, wherein said cell is a mammalian cell.
 15. An isolated nucleic acid molecule which encodes a tumor rejection antigen expressed in melanoma cells, wherein the complementary sequence of said isolated nucleic acid molecule hybridizes to the nucleotide sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 10 at 0.1×SSC, 0.1% SDS for 30 minutes at 65° C.
 16. An expression vector comprising the isolated nucleic acid molecule of claim 15, operably linked to a promoter.
 17. A host cell transformed or transfected with the isolated nucleic acid molecule of claim
 15. 18. The host cell of claim 17, wherein said cell is a fibroblast.
 19. The host cell of claim 17, wherein said cell is a mammalian cell.
 20. An isolated cDNA molecule which encodes a tumor rejection antigen precursor expressed in melanoma cells, wherein the complementary sequence of said isolated nucleic acid molecule hybridizes to nucleotides 2598-3542 of SEQ ID NO: 9 at 0.1×SSC, 0.1% SDS for 30 minutes at 65° C.
 21. An isolated cDNA molecule which encodes a fragment of a tumor rejection antigen precursor expressed in melanoma cells, wherein said fragment is processed by cell to a tumor rejection antigen, wherein the complementary sequence of said isolated nucleic acid molecule hybridizes to nucleotides 2598-3542 of SEQ ID NO: 9 at 0.1×SSC, 0.1% SDS for 30 minutes at 65° C.
 22. An isolated cDNA molecule which encodes a tumor rejection antigen expressed in melanoma cells, said tumor rejection antigen consisting of an amino acid sequence that is part of a tumor rejection antigen precursor, wherein said tumor rejection antigen precursor is encoded by a nucleic acid molecule the complementary sequence of which hybridizes to nucleotides 2598-3542 of SEQ ID NO: 9 at 0.1×SSC, 0.1% SDS for 30 minutes at 65° C.
 23. An isolated nucleic acid molecule which encodes a tumor rejection antigen precursor expressed in melanoma cells, encoded by nucleotides 2598-3542 of SEQ ID NO:
 9. 24. An isolated nucleic acid molecule which encodes a fragment of a tumor rejection antigen precursor expressed in melanoma cells, that is encoded by nucleotides 2598-3542 of SEQ ID NO:
 9. 25. An isolated nucleic acid molecule which encodes a tumor rejection antigen expressed in melanoma cells, the amino acid sequence of which consists of an amino acid sequence that is a fragment of the amino acid sequence encoded by nucleotides 2598-3542 of SEQ ID NO:
 9. 